MCS Midterm

Lysosomal Enzymes (Acid Hydrolases)

- addition of manose 6 and further phosphorylation man-6 will signal the protein to be sent to and degraded in the lysosome

Single gene defect

- defect involving a single gene; - mostly inherited - 1 - 1.5% of live born neonates - e.g. Cystic fibrosis, Sickle cell disease

chromosomal defect

- defect involving one or more chromosomes; - 0.5% of live born neonates - e.g. Down syndrome; 1/800 live births

I - cell disease (lysosomal storage disease)

- deficiency to phosphorylate mannose - worst kind of lysosomal disease - typically fatal - no degradative enzymes packaged in lysosomes and all lysosomal enzymes are secreted - overburden amount of stuff is stuck in the lysosomes - hirsutism, delayed developmental, kyphotic posture, flexion contractures of fingers, hips and kneeds, c

Triplet Genetic Code

- degenerate (64 codons for 20 amino acids) - The nuclear and mitochondrial genetic codes are similar but not identical - Boxes indicate the four codons which are interpreted differently - Degeneracy of the genetic code often involves the third base of codon - For e.g., GGN = glycine, CCN = proline, where N is any base

Homoplasmy

- term used to describe a mammalian cell whose copies of mitochondrial DNA are all identical.

Mesenchymal cells

- the ancestors of most native cells of adult connective tissue inset slide 20

cyanosis

- the appearance of a blue or purple coloration of the skin or mucous membranes due to the tissues near the skin surface being low on oxygen

Normochromic Anemia

- the concentration of hemoglobin in the red blood cells is within standard range. - However, there are insufficient numbers of red blood cells

Cri-du-chat

"5p-" deleted region on the short arm of chrom 5 microcephaly, delayed development, low birth weight, weak muscle tome, distinctive facial features, wide set eyes, low set ears, small jaw, rounded face, some have heart defect

Sickle Cell Incidence in the USA

- 1 in 12 African Americans carries trait - 1 in ~500 African Americans has sickle cell disease - 1 in every 36,000 Hispanic Americans - Total # with disease in the US is approximately 80-100,000

SCT/ Sickle Cell Disease Problems

- 1-5% mortality: New emphasis on considering transplant before end organ damage occurs - Graft versus host disease - Graft failure- 10% - Sibling matches are available in <20%: Push to collect umbilical cords of new siblings New multi-center trial for matched unrelated donors

Management of ACS

- Antibiotics - Oxygen if hypoxic - Albuterol and steroids if wheezing/asthma - Transfusion of PRBCs-packed red blood cells (simple or exchange) - Conservative fluid management - Incentive spirometry, ambulation - Pain medication

Sickle Cell Pathophysiology: Contributing Factors

- Blockage of small capillaries by sickle cells (vaso-occlusion) - Endothelial activation and damage - Adherence of WBC to endothelium - Platelet and coagulation factor activation

Hydroxyurea

- Developed as chemotherapy agent over 50 years ago to treat chronic leukemias - First use in SCA was 1984, approved by FDA in 1998 - Taken by oral route - Promotes switch to production of Hgb F - Dose limiting toxicity is myelosupression (bone marrow suppression - decrease in cells responsible for providing immunity, carrying oxygen, & those responsible for normal blood clotting)

History

- Discovered by John Langdon Haydon Down (1828-1896) 1866 - "Observations on an ethnic classification of idiots," London Hospital Reports 1909 - Association with advanced maternal age 1930s - Thought to be due to chromosomal error 1959 - 3 chromosomes #21

Summary of DS

- Even w/ increased focus on prenatal screening, DS will remain common - Delayed fertility may make DS more common - Early emphasis is on family support and managing life-threatening complications - Focus is replaced by optimizing health and educational attainment - Outlook for children and adults with DS have improved dramatically over the years, mostly due to emancipation of and advocacy by families affected by DS

Possible Sickle Cell Genotypes

- Hgb AS (sickle cell trait) - Hgb SS (sickle cell disease-SS, sickle cell anemia) - Hgb SC (sickle cell disease-SC) - Hgb Sb thalassemia - Hgb S/other e.g. SE, SG, SO, SD

Linkage for known disorders vs gene identification

- If the locus is known, one can look for flanking markers, and follow an allele through generations and in various relatives - If the locus is unknown, obviously linkage studies to help families is not really possible - One has to first identify where the gene maps, and ideally also identify the disease-causing mutation in that gene (once identified)

Complications of DS

- Individuals with Down syndrome almost universally have developmental and growth delay and hypotonia - Other complications as cardiac, gastrointestinal and hematologic occur with less but considerable frequency - These complications are thought to occur because of effects of 'gene dosage'

Risk Factors for Stroke

- Prior symptoms - Low steady state hemoglobin - High WBC - High blood pressure - Acute Chest Syndrome - Abnormal transcranial doppler

Diagnosis of Down syndrome

- The diagnostic test is the karyotyping of fetal cells, usually obtained by amniocentesis - Chorionic villus sampling may also be used - Postnatally, karyotyping of lymphocytes from peripheral blood is usually done - Rapid diagnostic tests are available for emergency situations, prenatally and postnatally - Fluorescent in-situ hybridization (FISH): Such result should be followed by routine karyotyping

Advanced maternal age

- The risk increases in a gradual, linear fashion until about age 30 and increases exponentially thereafter - For example, the risk of having a live born with Down syndrome is (Newberger, 2000): 25-year-old woman: 1/1,300 35 year old woman: 1/365 45 year old woman: 1/30

Effect of Maternal Age

- The risk of having a child with Down syndrome increases with advancing maternal age (Penrose, 1933) - Applies to non-disjunction in both meiosis I and meiosis II, but effect is greater in meiosis I

Genetic map of the human genome using linked, highly-polymorphic markers were created in the human genome project

- Today, we don't really uses markers a whole lot - we have started using SNPs more

Mendelian disease

- caused by mutation in a single gene - A person with that Mendelian disease has the gene mutation

ischemia

- restriction of blood flow to the tissues (due to lack of oxygen or blockage)

Organ Damage in Sickle Cell Disease

- retinopathy, stroke, pulmonary hypertension, acute chest syndrome, functional asplenia, hyposthenuria

euploidy

number of chromosomes is a multiple of 23 (n)

3 branches of cytogenetic mutations

numerical, structural, and other

Nucleus

principal site of DNA and RNA synthesis

Warburg effect

"aerobic glycolysis" Cancer cells only undergo glycolysis

Benign Tumors

"oma" not invasive or will not metastasize

p53

"the guardian of the genome" key in making repairs to damaged genome

95

% of those affected with down syndrome due to non-disjunction

1

% recurrence of down syndrome in couples where mother is under 35

diploid

(2n) in somatic cells

Beckwith-Wiedemann syndrome due to CDKN1C mutation

- dark colored - affected - light colored - carriers - those affected in next generation

Post-Translation Processing in the Secretory Pathway

- Removal of signal sequence - Partial proteolytic cleavage - Folding (and disulfide bond formation) - Glycosylation - Amino acid modification

Stratified columnar

- surface cells are columnar shape

Nuchal Translucency Thickness

- tendency for fetuses w/ Down syndrome to have increased fluid around the neck

Malignancies

(cancers) "carcinoma" Invade surrounding tissue and will metastasize

haploid

(n) in germ cells

BOTTOM LINE: You need to know:

* The basis for the Michaelis-Menten model of kinetics. * How to obtain Vmax and Km values from plots. * The major modes of enzyme inhibition. * The importance and basis of allosteric effects on activity. * The levels of regulation that can control the amount of enzyme activity. * The utility of enzymes for diagnostics.

Association studies and linkage disequilibrium

- ApoE alleles associated with Alzheimer disease - HLA-DR4 in 78% of Rheumatoid arthritis patients as opposed to 36% of the general population (UK)

Glucose 6-phosphate dehydrogenase (G6PDH) deficiency (STOP SIGN)

- (400 million afflicted worldwide; 10% African Americans, 13% Saudis, 60% Kurdish Jews), results from numerous missense mutations (at least 400 variants) of this X-linked gene. - Mediterranean allele F188 change to S. - In blacks, D376 to a N - Results in an unstable protein with shorter half-life. In red blood cells, which don't make new protein, the mutant enzyme (A- variant) half-life is 13 days vs. 63 days for wild type. - CELLS HAVE LOW AMOUNTS OF NADPH AND GLUTATHIONE TO COUNTERACT OXIDATION PRODUCTS.

2 Scenarios of Disease of Defective Collagens

- 1). Bad gene is a null or nonsense mutation (and the defective polypeptide is degraded quickly): 50% of the the normal collagen fibers are made - patient outcome: mild to moderate disease. - 2). Bad gene is a missense mutation in which the defective polypeptide does not fold correctly but still incorporates into collagen fibers: 3/4 of all of the fibers are bad since poisoned with the faulty subunit - patient outcome: severe to lethal disease. DOMINANT!

Duchenne Muscular Dystrophy(DMD)

- 1/3,000 male births, 1/3 result of new mutation - Frequently assoc. with intellectual impairment - Muscle degenerative disorder - Normal muscle breakdown with exercise is not repaired due to lack of structural protein - < 1% dystrophin - Affected boys are normal at birth, but develop progressive limb girdle weakness, calf hypertrophy, contractures, and cardiomyopathy - Usually in wheelchair by age 10-12 yrs, fatal by late teens early 20s - associated with near complete absence of dystrophin - <1% muscle fibers have normal dystrophin those may be the result of back mutations CK massively elevated at birth prior to development of symptoms

Results of STOP trial

- 10 CVAs in observation pts vs. 1 in transfusion pts 92% difference ( p < 0.001) Study terminated early

Beckwith-Wiedemann syndrome Laboratory testing - Cytogenetic

- 11p15.5 - Occasionally anomalies can be seen on high resolution chromosome analysis: Translocation, inversion, duplication in <1% Microdeletion or microduplication IC1 or IC2, or both - If DD is present, a cytogenetic anomaly is more likely

Cellular Membrane Proteins

- 20-residue stop-transfer sequences - Arrest passage across the ER, become embedded in the membrane - Internal signal sequence and stop sequence - Double-pass membrane protein

Muscular tissue

- 3 types - specialized for contraction - striated or non-striated

Impact of genetic disease

- 3% of all live born neonates have a significant genetic disease - 20% of all causes of infant mortality in the U.S. - 5 - 10% of all pediatric hospital admissions have a clearly defined genetic disease - Patients with genetic diseases are admitted more frequently and for longer periods

Nuclear Pore

- 3000-4000 pores throughout the nuclear membrane regulate entry into and exit out of the nucleus - Freely permeable for small molecules

Hemoglobin

- 4 subunits (each look like Mb) => 4 Hemes => 4 oxygens can be bound; 2 alpha subunits and 2 beta subunits. - Alpha and beta strongly interact via nonpolar patches to form a alpha-beta dimer. - A pair of dimers are held together by ionic and H-bonds. - Oxygen binding by Hb is variable and depends on the quaternary structure of the Hb tetramer, which in turn depends on its environment.

Triplet Repeat Expansions - Loss of function - differs between genes

- 5' non-coding, transcriptional suppression - 3' non-coding, abnormal RNA processing - intron

Directionality of DNA Strands

- 5'=> 3' - phosphodiester bonds joins 3' to 5' carbons of adjacent sugar molecules - Chargaff Rule: A-T & C-G base pairing - DNA: %A=%T, %C=%G;

Autosomal Dominant

- 50% of offspring of affected individuals are affected - Equal numbers of males and females affected - Vertical transmission - Unaffected members of a family do not have affected children - Variable expression is common - Decreased penetrance is possible - Isolated cases likely due to new mutation - Tuberous Sceloris Complex, Neurofibromatosis I, Marfan Syndrome, & Achondroplasia

DNA methylation at CpG dinucleotides

- 70% of CpG sites in the human genome are methylated - 15% of the CpG sites are concentrated in unmethylated CpG islands

Ubiquitin

- 76 amino acid protein - Covalently attached to proteins - Ubiquitinated proteins are targeted to the proteasome and degraded

Gene structure - function: variations on the general theme

- >50% of human genes show alternate splicing - Differential gene expression - genes are expressed in specific tissues and at specific developmental stages - A handful of genes are regulated by genetic imprinting - X chromosome inactivation: males and females have only one active X chromosome per somatic cell

Alternate splicing

- >50% of human genes use different combinations of exons to produce more than one type of protein

platelet adhesion

- A platelet receptor protein recognizes collagen of subendothelial cells via von Willebrand factor protein intermediate (if deficient, von Willebrand disease - one of the most common genetic bleeding disorders).

Alternate splicing (picture)

- A single gene may use different combinations of exons producing more than one type of protein - May be tissue specific and / or developmental stage specific - >50% of human genes show alternate splicing - Differential RNA processing results in tissue-specific products of the calcitonin gene

Nuclear DNA is packaged into chromosomes

- A small portion of the genome encodes proteins - Directionality of DNA strands and its consequences - Anatomy of human chromosomes

Unstable Hb (ex. Hb Koln).

- A substitution in normally nonpolar core causes precipitation (core turns inside out because of an improperly packed residue or an internal hydrophilic residue). - Such defects lead to Heinz bodies (Hb precipitates on the cell membrane) and hemolytic anemia.

Mutated gene for FAP

- APC

Biomedical Implications of degradation

- Accumulation of misfolded proteins and abnormalities in the degradation of intracellular proteins are associated with several diseases: Alzheimer's (and other neurodegenerative dis.) Alpha-1-antitrypsin deficiency - Proteasome Inhibitors as cancer therapeutic agents: Ubiquitin-Proteasome System is involved in the regulation of cell growth

Lysosome

- Acidic environment (pH≈5) - Contains multiple digestive enzymes (lysosomal hydrolases, at least 40) - Degrades proteins, nucleic acids, carbohydrates, and lipids - Substances from extracellular (endocytosis, phagocytosis, pinocytosis) and intracellular sites (autophagy)

BPG - O2 DELIVERY (Oxygen Delivery during Pregnancy)

- Adult Hb binds BPG well, but fetal Hb has a Ser residue substituted for a certain His residue, therefore BPG binds less tightly. - BPG is not able to lower oxygen affinity of fetal Hb. - differences in Hb affinity result in net oxygen transfer from mother (low affinity) to fetus (high affinity).

Collagen Roles

- Aging: - Cell Adhesion: - Wound Healing

Hardy-Weinberg Equilibrium

- Allele frequencies do not change from generation to generation - For any genetic locus, the genotype frequencies can be determined by the relative frequencies of the alleles at that locus

Alpers syndrome and other POLG syndromes

- Alpers: Severe disease of childhood - neurodegeneration, liver disease, due to AR mutations in POLG (mt polymerase gamma) causing mtDNA deletions and depletion - SANDO - sensory ataxia, neuropathy, dysarthria, ophthalomoplegia - Progressive External Ophthalmoplegia (PEO)

Biomedical Implication-Protein Folding:alpha-1 antitrypsin deficiency

- Alpha-1 antitryspin (αAT): protease inhibitor produced in the liver - Disease causing gene alterations result in abnormal folding of the protein: Decreased activity Failure to be normally secreted from the cell Accumulation within the cell

Hereditary Pancreatitis

- Alteration in the gene coding for trypsin can lead to activation within the cell and organ injury - Pancreatic inflammation (Pancreatitis) due to the destructive nature of activated trypsin within the pancreas

MICHAELIS-MENTEN MODEL

- An enzyme binds a substrate, converts it into product, and releases the product. - The enzyme is unchanged by reaction and performs multiple rounds of catalysis. Reaction Pathway: E+S=>ES=>E+P

Thrombin Negative Control

- Antithrombin III and heparin inhibit thrombin when it strays too far from clot site. - These inhibitors prevent clotting from wandering away from the wound site with activated platelets.

Exome sequencing

- Approach: collection of a cases of a certain disorder: selection of most distinctive / representative cases - Whole exome sequencing on a selected case - Sifting through and filtering of all SNPs identified - Aiming for SNPs that cause 'damaging' mutations, and segregate according to pedigree - Used for id'ing new genes & unknown diseases

Cytogenetics

- Approximately 95% of cases result from non-disjunction during parental meiosis - DNA molecular techniques can elucidate whether the additional chr21 is paternal or maternal in origin, and also whether non-disjunction occurred in meiosis I or meiosis II - The non-disjunction was found to be maternal in 95% of cases Maternal non-disjunction was found to be during meiosis I in 77% of cases

BPG - O2 DELIVERY (blood transfusion)

- At first, when storage for blood transfusions started, BPG was lost (0 mM) and caused problems for patients after transfusion. Hb without BPG had a high O2 affinity and was an "oxygen trap" that compromised sick patients. - Now add inosine to refrigerated blood to keep BPG levels up

Michaelis-Menten Plots: V vs [S]

- At fixed enzyme concentration [E], V is proportional to the substrate concentration, [S], at low [S]. - At high [S], V is nearly independent of [S] and dependent on [E].

Huntington Disease (Triplet Repeat Expansions)

- Autosomal dominant neurodegenerative disease - Variable onset - Adult onset; mean = 40y is typical - Juvenile HD <20y (severe) - Onset >50y (mild disease) - Slowly progressive, death 15-20 y from onset Clinical manifestations: - Involuntary movements - Chorea seen in 90% - Progressive dementia - Psychiatric disturbances - Anticipation

Epithelial Traits

- Avascular layers of cells - oxygen and nutrients diffuse to epithelial membranes from capillaries in adjacent connective tissue - Functions: protection, absorption, secretion, excretion and sensory

Answer to the Question

- B: enhanced

Fine-Tuning of O2 delivery (BPG - Negative Allosteric regulation) (

- BPG LOWERS Hb's ABILITY TO BIND O2 - Negative-charged BPG participates in an ionic bond network between beta subunits (His, Lys, amino termini) when in Taut deoxy form. - Normally, BPG is found in red blood cells at equimolar concentration (~5 mM) w/ Hb. - Under hypoxic conditions, such as lung disease, severe anemia, high altitude adaptation, BPG concentration increases (~8 mM) which enhances O2 unloading in tissues.

Systems biology integrates molecular biology

- Biology an information science (genome as"digital information" + environment) - Levels of information within a cell:genome → transcriptome → proteome - Levels of system complexity:molecules → cells → organs → individuals → ecosystem - Global,integrated,dynamic analysis is key

Uniparental disomy

- Both members of a pair of chromosomes inherited from the same parent - overexpression of genes from one parent - absence of parent specific transcript - initial meiotic non-disjunction - post-zygotic loss of third chromosome - loss of trisomic cell line, "trisomy rescue"

Mitochondria also important in most major metabolic pathways

- Build, break down, recycle cellular molecules - Enzymes for pyrimidine biosynthesis and heme synthesis - In liver, detoxify ammonia in urea cycle - Cholesterol, estrogen, testosterone synthesis - Neurotransmitter metabolism - Free radical production and detoxification

The folding of the polypeptide chain is constrained by steric hindrance and local bonding networks.

- Bulky R-groups allow only certain angles of rotation around the alpha-Carbon; likewise, charge attraction and repulsion of side chains can limit the range of available angles. - The polypeptide backbone therefore folds with a certain orientation and twist. - weak bonds will form that stabilize a particular structure like alpha helix or beta sheet. - all of the peptide bonds are H-bonding in these structures!

Treatment of Poisoning (CO)

- But since reversible gas binding to heme, you can utilize a high concentration of O2 (oxygen mask unless victim is in coma or pregnant then use hyperbaric treatment) to eventually displace CO (half-life of CO loss = ~9 hours w/o treatment to ~90 min w/ O2). - CO levels: 10% headache; 20% nausea; 30% mental function & vision deteriorate; 40-50% cardiovascular collapse, seizure, coma; 80% fatal. - Typical levels of CO:Hb in blood from smoking, 5-15%. - GIVE LOTS OF O2 TO DISPLACE CO FROM HEME OF THE Hb..

CAG expansion in the huntingtin gene

- CAG triplet repeat in exon 1 encodes a polyglutamine tract - CAG triplet repeat is highly conserved in evolution - Human = 18 - Mouse = 7 - Pufferfish = 4 Normal genes: 6 - 35 (median = 18) HD genes: 36 - 121 (median = 40) - Polyglutamine (polyQ) tract causes disease (NOT the CAG repeat per se) - Triplet repeat length correlates with disease severity

Mitochondria

- Contains circular dsDNA called mtDNA - Powerhouse of the cell: ETC produces ATP Contains complexes I, II, III, IV, V All but complex II contain at least one subunit coded for on the mtDNA - Different cell types have different numbers of mitochondria

Poisoning (Cyanide-CN)

- CN binds to methemoglobin (1% of Hb normally) but not Hb. - To protect mitochondrial electron transport after poisoning, treat with amyl nitrite to make a little more MHb; this form of Hb will trap CN in blood. Hazards (burning rubber or plastic, metal plating chemicals or photography, some plant ingestion, banned solvents, or nail polish removers)

Fine-Tuning of O2 delivery (CO2 levels-Negative allosteric regulation) (How can Hb sense and transport CO2, too)

- CO2 LOWERS Hb's ABILITY TO BIND O2 - CO2 covalently binds to N terminus of Hb chains (creates a negative charge) to form carbamylHb - Lower O2 affinity (positive residue interacts with negative carbamate group in an ionic bond=>Taut form is stabilized). - Enhancement of O2 delivery in tissues at work plus removal of some CO2 from tissues (most CO2 by other mechanisms).

Cartilage

- CT specialized for structural support. - Flexible and compressible. - Forms fetal skeleton. - Avascular, no lymphatics or nerves. - Receives nutrients via diffusion. - Composed of chondrocytes and extracellular matrix. - Chondrocytes located in lacunae.

Enzyme inhibition

- Certain molecules, inhibitors (I), can stop or slow catalysis by binding to the active site or regulatory site of enzyme. - This is THE major mechanism of action of most commonly used drugs. - Types: irreversible inhibition & reversible inhibition (competitive, non-competitive & uncompetitive inhibition-NOT MCS),

Increased Affinity (Abnormal O2 transport)

- Change in alpha/beta contacts of Hb elicit changes in the Relaxed/Taut ratio. - This problem leads to polycythemia; 20% more Hb per cell and more red blood cells/ml are made to compensate for poor oxygen delivery characteristics

Epigenetic Modification of the genome

- Chemical modification of histones determines transcriptional activity of genes - this represents functional organization of the genome - DNA is methylated at CpG dinucleotides

Epigenetic modification of the genome

- Chemical modification of histones determines transcriptional activity of genes - this represents functional organization of the genome - DNA is methylated at CpG dinucleotides

Types of genetic disease

- Chromosomal defect - Single gene defect - Multifactorial/polygenic disease - Mitochondrial disorders

Anatomy of Human Chromosomes

- Chromosomes are morphologically classified based on the location of the centromere - "p" (petite) = short arm; "q" = long arm - The kinetochore is present at the centromere; site for microtubule spindle attachment for chromosome segregation during cell division

CPEO=PEO (genetically heterogeneous

- Chronic Progressive External Ophthalmoplegia - Unable to move eyes fully in all directions (Note this patient also has eyelid ptosis or drooping) - Can be multiple mtDNA deletions (sporadic) - Can be mtDNA point mutations - Can be AD and AR mutations causing mtDNA deletions - Can be neuromuscular and other symptoms

A large portion of the genome is repetitive DNA

- Classes of repetitive DNA - Tandem repeats are often polymorphic - Interspersed repeats may undergo transposition - Epigenetic modification and genome defense

Signal Peptidase

- Cleaves Signal sequence prior to completion of translation - located on the inner surface of the ER membrane

Effect of Mutations in a Multimeric Protein

- Collagen is a triple helix than can be made of more than 1 species of polypeptide (depending on the fiber and the tissue). - There are 2 alleles of each collagen gene species (remember ~28 families) per diploid cell.

Ex - bacterial aspartate transcarbamoylase with 6 catalytic subunits + 6 regulatory subunits.

- Committed step for feeding into pyrimidine pathway - ATP is 2 steps upstream in pathway - positive effector - CTP is a final pathway product - negative effector

Glycosylation

- Common chemical modification of secreted & membrane bound proteins - 2 general types: N-linked - attachment through the amino (-N) group of the side chain of asparagine O-linked - attachment via the hydroxyl (-OH) group of serine or threonine Simple or complex with branching networks of 1000 sugars NO NEED TO MEMROZIE

Phosphorylation

- Common protein modification (Serine, Threonine, Tyrosine (1000:100:1)) - Serves to regulate the biologic activity of a protein (transient, reversible) or to target to a location within the cell - Kinases - enzymes that phosphorylate proteins - Phosphatases - enzymes that remove phosphates - there can be many different pattterns

E-aminocaproic acid [EACA] and tranexamic acid

- Competitive plasmin inhibitors can be used to slow down clot removal by inhibiting fibrin digestion. - During dental surgery and neurosurgery, the prophylactic use of such drugs is particularly helpful. - However, extreme caution must be observed to avoid systemic coagulation in the fine capillaries and organs. (Keep some heparin ready and available!).

Mitochondrial diseases: extremely complex

- Complex biochemistry and clinical metabolic profiles - Complex relationship between nuclear and mitochondrial DNAs and proteins they encode - Progressive disease, single or multisystem dysfunction

Heterochromatin

- Condensed chromatin - Deacetylated & methylated histone tails - Methylated DNA - Seen at repetitive DNA (defense)

Genetics of multifactorial / complex diseases

- Coronary artery disease - Schizophrenia - Autism - Diabetes mellitus - Multiple sclerosis - Epilepsy - Rheumatoid arthritis - Alzheimer disease - Congenital malformations - Morbidity/mortality in 2 out of 3 people in their life-time

irreversible inhibition

- Covalent complex of inhibitor and enzyme. - Inactivates enzyme by a reaction of the I with a critical site. (ex. an acetyl group from aspirin reacts with cyclooxygenase; organophosphate nerve gas & pesticides react w/ reactive serine of acetylcholinesterase)

Genetics of multifactorial / complex diseases (Traits)

- Cumulative effect of multiple genes and their interaction with the environment - Polygenic traits are continuous and quantitative (e.g. blood pressure, height) - Quantitative trait loci (QTL) and disease susceptibility loci

Stem Cell Transplant in Sickle Cell Disease

- Currently the only "cure" for sickle cell disease - In the past we would consider transplant only for :- Patients who had had a stroke Patients with repeated episodes of acute chest Patients with very frequent VOC (heart defect) and dismal quality of life

Proteasome

- Cylindrical structure lined with proteases - Degrades ubiquitinated proteins - Present in the cytoplasm and nucleus - Constitutes 1% of cellular protein

Nuclear Hormone Receptors

- DNA binding proteins (z Fingers) - Dimers - Conformational change upon ligand binding

DNA Replication

- DNA replication is semi-conservative - DNA is replicated in the 5' to 3' direction (3' end) - Directionality of DNA strands  - The two strands of DNA are replicated in opposite directions

Translation

- Decoding of messenger RNA (mRNA) by the ribosome to produce a chain of amino acids (polypeptide) - Synthesis of a protein under the direction of the mRNA

Imprinting

- Dependent upon parent of origin: expansion of triplet repeats & phenotypic expression - Disorders: Triplet repeat expansion: anticipation Myotonic dystrophy Huntington disease Fragile X syndrome Epigenetic modifications: genomic imprinting gene silencing Prader-Willi/Angelman syndromes

Protein Signal Sequence

- Determines the site of protein synthesis - Free-cytoplasmic or ER-bound ribosomes - 20-25 amino acids (mainly hydrophobic) - Amino (N-terminal) end of the polypeptide chain (first amino acids synthesized in a polyptide)

Gene Imprinting

- Differential gene expression based on parent-of-origin - The repressed / inactive gene is said to be "imprinted" - Caused by DNA methylation and altered chromatin - Imprinting may be tissue-specific

Gap Junctions

- Direct connection between cytoplasm of 2 cells (electrical/ chemical integration) - Opening is regulated - Passable for molecules up to ~1,000 Dalton - Occurrence: everywhere, but especially: Heart muscle (signal to contract) & Many neurons in retina

Multifactorial/polygenic disease

- Disease caused by interaction of multiple genes +/- environmental factors - 1% of live born neonates - e.g. common congenital defects [NTDs, congenital heart disease

Cancer & environment

- Disease results from a combo genetic and environmental factors - pyramid between genetic, noninfectious environment and infectious

AAs with special features:

- Disulfide link - crosslink two cysteine (C) groups to join polypeptides together (can be held up covalently) - Glycine - G - a hydrogen side chain, small, thus no steric hindrance; can pack tightly. - Proline - P - a secondary amine with a ring; kinks polypeptide chain (can be used to make globular protein)

Human X Chromosome

- Divided into distinct regions by its banding pattern - G-banding: most widely used technique for studying mammalian chromosomes - International System for Cytogenetic Nomenclature - Arm, region, band, sub-band e.g. Fragile X syndrome - "Xq27.3" means long arm of X, region 2, band 7, sub-band 3

Sickle Cell Disease - New Strategies

- Drugs to promote Hgb F e.g. Hydroxyurea - Stem Cell Transplant - Gene Transfer Therapy

Elastic cartilage

- Elastic fibers (elastin) in matrix. - Also has some Type II collagen. - Chondrocytes very abundant. - External ear. - External auditory meatus. - Eustachian tube. - Epiglottis. - Perichondrium present.

elastic

- Elastin & fibrillin - branched and distensible

Laboratory evidence of CFTR abnormality

- Elevated sweat Cl- concentration (≥ 60 mmol/L) on two separate occasions • by pilocarpine iontophoresis / NCCLS guidelines • interpretation of sweat chloride results - Identification of two CF-producing mutations - Abnormal "CF pattern" with Nasal Potential Difference Measurement

"Pink Puffers"

- Emphysema is an enlargement of the air spaces distal to the terminal bronchioles, with destruction of their walls. - word used to describe People with emphysema due to their skin complexion

New Screening for Sickle Cell Disease

- Evidence for early identification and treatment: Penicillin in Sickle Cell Studies - All 50 states now provide mandatory universal newborn screening - Specimen drawn prior to transfusion

Glycosylation Intra & extra

- Extracellular & Membrane-bound proteins are frequently modified by glycosylation - Intracellular proteins are infrequently modified by glycosylation except for O-GlcNAc addition (ying/yang to Phosphorylation)

Enzyme-linked Cell Surface Receptors

- Extracellular ligand binding domain - One transmembrane segment (per subunit) - Cytosolic domain with intrinsic enzymatic activity (or associated with enzyme) 1. Receptor tyrosine kinase (RTK) 2. Tyrosine kinase-associated receptor 3. Receptorlike tyrosine phosphatase 4. Receptor serine/threonine kinase (TGF-beta) 5. Receptor guanylyl cyclase (Natriuretic peptides)

Loeys-Dietz Syndrome

- Facial dysmorphism - Scoliosis -Aortic dilation (aneurysm, dissection) - Translucent skin - Flat feet - TGFBR1 or TGFBR2 mutations - Similar to Marfan syndrome, where mutations in extracellular Fibrillin-1 affect sequestration of TGFb

Extrinsic pathway

- Factor VII and a subendothelial membrane protein tissue factor combine to form a complex that converts X to Xa - Usually extrinsic pathway is sufficient for hemophiliacs (missing VIIIa of intrinsic pathway) to combat spontaneous hemorrhages except in tissue factor-poor areas like joints and muscles (these continual bleeds lead to arthritis).

Genes vs Environment

- Familial aggregation and relative risk (empiric risk of recurrence) - Twin studies (concordance vs discordance)

Characteristics Twins

- Familial aggregation with no Mendelian inheritance - Disease is more common in close relatives and becomes less common with decreasing relatedness - Reduced penetrance: MZ twins share all genetic predisposing factors but environmental factors may differ and concordance is <100% - Population-based empiric risks are used to estimate recurrence - Recurrence risks may be modified based on other factors

Protein conformation (particular spatial arrangement of atoms and bonds of a protein.)

- Fragile structures (ΔG ~5-20 kcal/mol). - Proteins can be denatured or killed by the loss of a few weak bonds: - Heating can denature by disrupting bonds (remember they are weak bonds, generally ~1-3 kcal/mol each). - pH changes can unfold (loss/gain of salt link or cause repulsion). - Solvent change (turn protein hydrophobic core inside out). - Toxic ligands (heavy metals can disrupt disulfide links).

2 Sites for Translation

- Free Cytoplasmic Ribosomes: proteins of the cytoplasm, nucleus, and mitochondria - Endoplasmic Reticulum-bound Ribosomes: secreted, plasma membrane, ER, Golgi apparatus, and lysosomal proteins

KRAS

- GTP-binding signal transduction protein - mutations can drive cellular proliferation - mutations can be detected through sequencing (limit of detection=5% mutant alleles)

Activation of G proteins

- Galpha: GTPases, GTP-bound (active), GDP-bound (inactive) - RGS proteins can be GTPase activating proteins - Galpha (dissassociates - GTP)

Fragile X (Molecular)

- Gap may be due to uncondensed DNA or altered chromatin structure - FMR1 gene - Triplet repeat expansion - usually >200 repeats - Leads to methylation in the promoter region - Loss of function - Hypermethylation - Maternal expansion

Michaelis-Menten Plots: V vs. [S] Vmax & Km

- Maximal velocity, Vmax - defined as the reaction rate where enzyme is saturated with substrate. - Michaelis constant, Km - defined as the concentration where velocity is ½ of Vmax.

primary structure is altered by gene mutation

- Gene duplication allows the new copy to mutate to a novel form that may play a (sometimes entirely) different role in the body. - Isoforms or isozymes are proteins with distinct sequences that perform the same general function. Optimize performance for functions in other tissues or developmental stages - Homology - Close similarity or regions of identity between protein sequences are often found.

Translating Dosage Compensation to Trisomy 21 (Article)

- Gene imbalance across an extra chromosome can be corrected by manipulating the XIST gene (X-inactivation gene)

Transfer of genetic information (the general theme)

- Gene structure and transcription - Transcripts of protein coding genes are processed - Splicing is directed by consensus sequences - The genetic code is degenerate (involving the 3rd position)

Differential gene expression

- Genes may be expressed only in specific tissues and developmental stages

Genomic Organization of genes

- Genes of different sizes are spread throughout the genome - Some genes are organized in clusters - It is not always possible to define a gene as a discrete unit - A large portion of the genome is transcribed on both strands!

Special forms of differential gene expression

- Genetic imprinting - X-chromosome inactivation

Dosage Compensation

- Genetic regulatory mechanism which operates to equalize the phenotypic expression of characteristics determined by genes on the X chromosome so that they are equally expressed in the human XY male and the XX female

Heat Shock Proteins (HSP)

- Glucocorticoid Receptor - Androgen Receptor - Estrogen Receptor

Fever

- Guidelines are standardized and available in every E.R. in the state. - Guidelines are sent to the PCP of every baby identified on newborn screening. - Evaluation must include: Blood culture Long-acting antibiotic Close follow-up - Guidelines should be followed even if the patient has another source of fever e.g. URI or otitis media

Variable (age-dependent) penetrance (HD)

- HD is autosomal dominant - Risk of recurrence is 50% - Many refuse presymptomatic testing - Significant variability in the age of onset - Risk of HD in first-degree relatives with a priori risk of 50% decreases as the person grows older

General Characteristicsof Epithelium:

- Highly cellular. - High regenerative power. - Exhibit polarity. - Surface cells rest on basement membrane. - CT below BM.

Heterochromatin

- Highly condensed, compact, transcriptionally inactive DNA - Constitutive - always inactive (1q, 9q, 16q) - Facultative - either active / inactive form (X inactivation)

Hyaline Cartilage

- Homogenous matrix - type II collagen - Surounded by perichondrium. - Growth occurs appositionally and interstitially. Fetal skeleton, Articular cartilage, epiphyseal plates, Costal cartilage of ribs, Nasal cartilages, Trachea, bronchi, cricoid and thyroid cartilage.

Limitations of homozygosity mapping

- Homozygosity mapping only applies to AR disorders - Such approach is not applicable to AD and XLR disorders - Modern approaches to gene identification in AD and XLR disorders: Exome sequencing Whole genome sequencing

PANCREATITIS

- INSTEAD OF TRYPSINOGEN BEING MADE IN THE PANCREAS AND TRANSMITTED INTO INTESTINE WHERE THE FOOD IS WAITING FOR IT, IT ACTUALLY POPS IN THE PANCREAS AND WILL START TO EAT THE PANCREAS..

Twins Reared Apart

- Identical genotypes in dissimilar environments - Efficient way to analyze conditions that have a strong environmental component - Alcoholism: similar concordance rates in MZ twins reared together or apart (i.e. shared genetics >>> shared environment)

Weak acid/bases can change charge as the environmental pH

- If amino group gains a proton [H+] (at low pH) => positive charge; if it loses H+ => neutralized. - If carboxylate group gains H+ => neutralized; if it loses H+ => negative charge.

Enzyme Kinetics

- Measurements of reaction velocity under various conditions helps reveal the mechanism of catalysis as well as assist the design and the of drugs to alter reaction rate.

Practical application of the Hardy-Weinberg Equation / Principle

- In AR diseases it is not possible to calculate the frequency of "carriers" by direct observation - HW equation / principle can calculate this if the disease frequency is known (for that population) - Population must be in HW Equilibrium for the calculation to be reliable (large population, random mating)

Proglucagon (STOP SIGN)

- In the pancreas, you chop off glucagon & MPGF - in intestine and brain, the oxyntomodulin, GLP-1, GLP-2, IP2 - NO NEED TO MEMORIZE THIS - JUST KONW THAT PROTEINS CAN BE MADE IN DIFFERENT WAYS IN DIFFERENT PARTS OF BODY

Fibrocartilage

- Intermediate of hyaline cartilage and dense regular CT. - Type I collagen bundles visible in matrix (also has Type II collagen). - Isogenous groups. - No perichondrium. - Articular disks, menisci, pubic symphysis, intervertebral disks, sites of insertion of ligaments and tendons into bone.

PIP3: Phosphatidylinositol-(3,4,5)-trisphosphate

- Is the product of the class I phosphoinositide 3-kinases (PI 3-kinases) phosphorylation on phosphatidylinositol (4,5)-bisphosphate (PIP2). - It is a phospholipid that resides the plasma membrane.

Triplet Repeat Expansions

- Islands in the genome containing three base pairs repeated over and over - Expansions are common cause of neurologic or neuromuscular disorders - Increased numbers of repeats cause malfunction: Gain-of-function & Loss-of-function - Other short tandem repeats of varying sizes may be pathogenic

Imprinting Means (Question)

- It matters that we inherit genes from both parents!!!

Fine - Tuning of O2 delivery (pH levels-Negative Allosteric Regulation-pH/protons) (Are those muscles burning with acid buildup after a work out?)

- LOW pH LOWERS Hb's ABILITY TO BIND TO O2 (BOHR EFFECT) - Upon oxygenation, protons are released from Hb (pKa changes in R/T forms; loss of ionic bonds). - Extra protons (derived from CO2 dissociation and lactic acid/metabolism) will restore ionic bonds thus cause Hb to release O2. - Enhancement of O2 delivery in tissues at work

Leigh Syndrome

- Lactic acidosis of blood and brain (varies) - White matter disease (leukodystrophy) brain pattern on MRI - Severe neurologic involvement/early death - Some cases respond to DCA (dichloroacetate), which stimulates Pyruvate Dehydrogenase in clinical trials - Multiple genetic etiologies: mtDNA, recessive nDNA, X-Linked genes

Copy Number Variations (CNV)

- Large segments of DNA (Large repeats or could also be deleted or duplicated) - 10 kb - 5 Mb - ~12% of the genome (n ~1500)

Velocardiofacial syndrome (more symptoms)

- Long face - Wide nose - Hooding of upper eye lids - Bulbous nasal tip - Hypernasal speech - Immune deficiency - Hearing loss - Psychiatric illness - Autosomal dominant - Chromosome 22q11.2 deletion

Types of Connective Tissue

- Loose connective - Dense connective (irrgeular and regular) - based on appearance of collagen fibers

Beckwith-Wiedemann syndrome Laboratory testing - Molecular

- Loss of methylation on maternal IC2 - 50% - Paternal uniparental disomy 11p15.5 - 20% - Gain of methylation on maternal IC1 - 5% - Mutations on maternal copy of CDKN1C - 40% of familial, 5-10% of new diagnoses - Red - maternal, blue - paternal,

Mutated gene for Lynch Syndrome (HNPCC)

- MLH1, MSH2, & others

Function of Telomere

- Maintaining the structural integrity of chromosomes - Facilitating replication of the ends of chromosomes (lagging strand synthesis)

X-chromosome inactivation in female mammals

- Males have one X and females have two X-chromosomes - Females inactivate 1 X-chromosom to equalize expression of genes located on X-chromosome (dosage compensation)

QUATERNARY STRUCTURE (complex of multiple polypeptides held together by various interactions)

- Many oligomeric proteins are bound by noncovalent, weak interactions and/or disulfide links. Some potential benefits of oligomeric proteins: a. Transmit information between subunits (e.g., hemoglobin). b. Mix and match functional units between various proteins (e.g., G-protein receptors). c. Pass a substrate directly to next "enzyme" in pathway because it is in close proximity in the complex (e.g., fatty acid synthase complex).

Duplication 22q11.2 syndrome

- Mild facial dysmorphism: Downslanting eyes Broad nose with bulbous tip Posteriorly rotated ears Mild micrognathia - Cleft palate - Congenital heart defect - Hydronephrosis - Seizures or infantile spasm - Intellectual deficits - Speech delay - Behavior or psychiatric problems

Post-translational modification

- Modification of a polypeptide after translation - Primary structure of a protein is not always sufficient to explain its biologic activity. - Polypeptides are inactive or nonfunctional as they exit the ribosome. Modification is often necessary for their activity.

Concordant Twin Studies

- Monozygotic (MZ) twins are genetically identical. - Dizygotic (DZ) twins, like sibs, share 1/2 their genes - If a trait is 100% genetic (0% environmental) then 100% of MZ twins and 50% of the DZ twins will manifest it (concordance) - If a trait is entirely non-genetic then both MZ and DZ twins will be equally concordant - In multifactorial diseases with a significant genetic component MZ twins are concordant much more frequently than DZ twins

Collagen Role - AGE

- More cross-linking of the lysines of collagen molecules with time, therefore fibers stiffen as one ages (old steer steaks tougher than veal!).

Collagen

- Most abundant protein (skin, cartilage, tendons, bone). About 25% of protein in adult & 15-20% of protein in child. - Family of at least 28 different genes.

Silver-Russell Syndrome (UPD chromosome 7)

- Most the result of Robertsonian translocation inherited from the father - Some paternal isochromosome 14 - Critical region 14q32 - Paternal isodisomy - Maternal isodisomy - bell-shaped thorax observed in neonate w/ upd 14 pat

Genomic Disorders

- Multiple malformations (major and minor) - Frequently MR, DD, behavior abnormalities - Widely variable phenotype - Some features may be inherited as Mendelian conditions

Mutated gene for neurofibromatosis

- NF1

Crystalline or polymeric Hb (Decreased solubility)

- New contacts between mutant Hb tetramers arise that allow polymerization and aggregation (ex. HbS Glu goes to Val) - Polymers/aggregates directly distort red blood cell shape and leads to microvascular occlusion. - deformed red cells are more readily destroyed under stresses of blood flow in vasculature resulting in hemolytic anemia.

Hydrophobic & philic

- Non-polar residues hide in the protein core or in lipid membranes. - Polar residues typically reside at the protein surface in contact with water

R-group substituents can be grouped into:

- Nonpolar side chains: F, A, L, I, W, V, M, P are C-H bond rich (greasy!). For the most part, these groups hide inside the protein core away from water (hydrophobic) - Polar side chains: like water (hydrophilic) and/or are charged: - Contain OH => S, T, Y; contain H2NC=O (amide)=>N, Q. - Acidic carboxylic acids can donate proton (become negatively charged) => D,E. - Basic amine can accept protons (become positively charged) => K,R,H.

Pinocytosis

- Nonselective uptake of fluid - Recycle the plasma membrane

DNA to Chromosmoe

- Nucleosome is the fundamental unit of DNA packaging - Eight histone proteins around which 146 bp of DNA is coiled 1.75 turns - Further coiled into a 30 nm chromatin fiber - Loops of chromatin fiber (~75 kb) are attached to a central scaffold - Further coiled to form the chromatid fibers seen in metaphase - Six feet of DNA in a single cell is compacted 10,000 fold

Proteolytic Cleavage

- Occurs in most proteins: Simplest form - removal of the initiation methionine present in most proteins Proteins secreted from the cell undergo cleavage - Many proteins are synthesized as inactive precursors - activation occurs by limited proteolysis under proper conditions

Glycoprotein Synthesis: N-linked

- Occurs in the lumen of the ER - Involves a lipid of the ER membrane (dolichol pyrophosphate) - Oligosaccharide is constructed on the lipid - Transferred to the protein by a protein-oligosaccharide transferase in the ER

Glycoprotein Synthesis: O-linked

- Occurs in the lumen of the Golgi - Oligosaccharide is constructed directly on the protein

Thrombin Production (step 2)

- On the platelet surface, factor Xa), a serine protease, cleaves prothrombin in 2 places. - Active thrombin falls off platelet (lipid binding domain cut off) and gets enmeshed in fibrinogen/fibrin network. - Thrombin now starts cleaving fibrinogen to form insoluble fibrin clot.

Imprinting of genes may be seen only in specific tissues or developmental stages

- Only a minority of genes are imprinted (~60) - Imprinting is predominantly seen in mammals

Euchromatin (Active)

- Open chromatin - Acetylated & unmethylated histone tails - No DNA methylation - At actively expressed genes (functional regulation)

PI3K acts through Akt (PKB)

- PIP3 is recognized by PH (pleckstrin homology) domains of Akt and PDK - PIP3 activates Akt & PDK to activate further downstream signals

Prader Willi Syndrome & Angelman Syndrome

- PWS = Paternal Deletion - AS = Maternal Deletion OF SAME REGION IN CHROMOSOME 15

Sickle Cell Pedigree

- Parents with sickle cell trait: hemoglobin AS - Probability of child with hemoglobin AA: 25% - Probability of child with sickle cell trait AS: 50% - Probability of child with sickle cell disease SS: 25%

"Blue Bloaters"

- People with advanced COPD that have primarily chronic bronchitis - word used to describe people w/ advanced COPD because of the bluish color of the skin and lips (cyanosis) along with hypoxia and fluid retention

Phagocytosis

- Performed by specialized cells - Plasma membrane engulfs extracellular substances - Internalized and fuses with lysosomes

Peptide bonds

- Planar peptide bond - The carbonyl electrons are delocalized and forms a rigid, partial double bond C-N bond. - Most (99.99%) in trans configuration.

clot retraction

- Platelets tighten clot by contracting a smooth muscle protein, called thrombosthenin (about 1 hour). - This final clot is very strong and stable

Genetics of multifactorial / complex diseases (Traits Cont'd)

- Polygenic diseases show familial aggregation - Genetic counseling in multifactorial diseases is based on empiric risks, which are modified by various factors - Twin studies are helpful in estimating the contribution of genetic determinants of disease

Beckwith-Wiedemann syndromeClinical diagnosis

- Positive family history, Macrosomia, Ant. linear ear lobe, post. helical ear pits, Macroglossia, Omphalocele, Visceromegaly, Embryonal tumor, Hemihypertrophy, Renal anomalies & Cardiomegaly - Polyhydramnios, prematurity, Neonatal hypoglycemia, Midface hypoplasia, Cardiac malformations, Diastasis recti & Advanced bone age - Overgrowth syndrome, tend to be large, unusual ear pits and ear creases

Acute Pancreatitis

- Premature activation of digestive enzyme zymogens in pancreas (pancreatic enzymes are supposed to be in the intestine). - The organ self-digests in a chain-reaction and releases digestive enzymes into abdominal cavity. - This disease is diagnosed by a laboratory test for pancreatic enzymes (e.g. lipase or amylase) in the bloodstream. - Gallstones, trauma, alcohol abuse, genetic mutations & smoking commonly instigate pancreatitis. Patient presents in much pain with a hard, distended belly, and shows the Grey Turner sign ("bruise").

Management/Intervention

- Prenatal diagnosis and management issues will be reviewed by Dr. Wagner - Postnatal issues: Establishing diagnosis - DS is a clinical diagnosis: Karyotyping/FISH testing to confirm and to rule out other causes than trisomy 21 Inform family, and arrange for support system, esp. if diagnosis not anticipated

BCL2

- Prevents apoptosis - use immunostain to look for high levels in cancer cells - intrinsic and extrinsic pathway (caspases)

trypsinogen

- Produced in the pancreas - Secreted into the intestinal lumen - Digestion of proteins in food

Signaling Molecules

- Proteins (EGF) - over 100 aa's - Peptides (glucagon) - less 100 aa's - Amino acids (glycine) - Nucleotides (cAMP) - Steroids (estrogen) - Fatty acid derivatives (arachidonic acid) - Ions (Ca2+) - Gas (NO) - Light - Secreted by exocytosis - Released by diffusion - exposed to Extracellular space - released through channels

MEDICAL RELEVANCE OF PROTEINS

- Proteins from the nucleus (e.g. histones, polymerases), cytoplasm (e.g. basic metabolic enzymes, parts of ribosomes, actin/myosin of muscle fibers), cell surface (e.g. receptors for hormones & neurotransmitters), and extracellular fluids (e.g. clotting factors & immunoglobulins) make human life happen! - Proteins serve as structural elements, catalysts, signaling molecules, and molecular machines that if not working properly, result in disease or death.

Localization (trafficking) of cytoplasmic proteins

- Proteins produced on free cytoplasmic ribosomes - Contain a sorting signal - Recognized by sorting signal receptor at target location - Examples: import into nucleus, mitochondria, and peroxisome

Nuclear Localization of Proteins

- Proteins produced on free cytoplasmic ribosomes - Nuclear Localization Signal: Patch of cationic amino acids (lysine and arginine) on the surface of the folded protein - NOT CLEAVED - Recognized by nuclear import factor: Importin-a - Importin-b transports NLS-importin-a into the nucleus through the nuclear pore - Energy-dependent process

Glycoproteins (structure & bonding)

- Proteins with covalently attached oliogosaccharide - Short carbohydrate chain as compared to proteoglycans: usually 2-15 residues - Chains of monomers - Linear or Branched chains - Not necessarily negatively charged - Cellular functions include: Cell surface recognition, antigenicity, extracellular matrix, mucins within the GI tract, almost all globular proteins in plasma Protein structure, solubility, and stability

Connective Tissue

- Provides structural and metabolic support for other tissues. - Highly vascularized. - Abundance of extracellular matrix with relatively few cells.

Interspersed Repeats (dispersed across the genome)

- SINES (Short Interspersed Nuclear Elements) ~10% of genome Alu repeats: primate-specific, approx. every 3 kb - LINES (Long Interspersed Nuclear Elements) ~10% of genome L-1 repeat: encodes a reverse transcriptase and can retrotranspose

Prenatal Screening and Diagnosis (Cont'd)

- Screening includes measuring multiple second trimester maternal serum markers - Asking a woman her age, and offering amniocentesis if she is age 35 or older is also considered a screening test - Other screening methods include fetal ultrasonography, looking for nuchal translucency thickness and/or absence of the nasal bone

Colon cancer screening removes pre-malignancies

- Screening rationale:some adenomas become invasive carcinoma,so remove - Age 50-75 • every 10 years:colonoscopy • every 5 years:flexible sigmoidoscopy • yearly:fecal occult blood testing

Acute Chest Syndrome

- Second most common reason for hospital admission - > 50% of patients have at least one episode during childhood - Patients with all genotypes may experience acute chest syndrome - Patients with repeated episodes are at high risk for chronic lung disease and early death

Receptor-Mediated Endocytosis:

- Selective uptake of extracellular substances - Requires binding to cell surface receptor - Important functions: Uptake of nutritive substances Waste disposal Mucosal Transfer

2 Types of Nuclear Hormone Receptors

- Sequestered by Heat shock proteins - Constitutively Nuclear

BRAF

- Signal transduction protein downstream of KRAS - can be inhibited w/ kinase inhibitor - mutations can also be detected using sequencing

MICHAELIS-MENTEN MODEL (How do you quantify an enzyme concentration?)

- Simplified assumptions - measure the velocity, V, of the forward reaction where: a) enzyme is limiting, b) substrate in excess, and c) reverse reaction is negligible at early time points (<5% of substrate is consumed).

Myoglobin

- Single polypeptide chain with 153 AA. - Compact globular protein with ~80% alpha-helix in 8 segments. - Protein interior is mostly nonpolar amino acids, major stabilization from hydrophobic effect. - Surface is mostly polar amino acids, thus highly soluble. - Heme in a pocket protected from water.

Phenotype variation in AR disease

- Some children have mild start then regress (boy, left 2), especially with infection, etc - Many children are severe from the beginning (right) - NOTE: can be differential expression of AR genes in different tissues, mimicking mtDNA heteroplasmy

Nuclear-encoded mitochondrial diseases - Classifications

- Some encode subunits of Complexes - Some encode proteins which assemble specific multi-subunit Complexes - Some encode protein defects causing instability in mtDNA: mtDNA Deletion disorders mtDNA Depletion disorders - Others - various functions

Telomere Structure and Function

- Specialized structure that caps the ends of chromosomes leaving no free DNA end - STRUCTURE: 10 - 15 kb repeated array of TTAGGG bound to a specific set of proteins - Telomere length is maintained by telomerase - Telomerase is absent in most normal somatic cells (present in germline) - Shortens 100 bp per cell division (eventually leading to senescence) - Telomerase is active in 90% of cancers

Consensus sequences for the splice donor and acceptor sites

- Splicing is a complex process directed by sequence elements at the 5' (splice donor) and 3' (splice acceptor) ends of introns - Endonucleolytic cleavage of introns and fusion of adjacent exons

Genomic Disorders Chromosomal microdeletion syndromes Contiguous Gene Syndrome

- Sub-microscopic deletions/ duplications of genetic material - Usually more than one gene involved - Areas of high recombination flanked by and due to unique low copy repeats - Following non-homologous recombination caused by misalignment of chromatids during meiosis

Major classes of Repetitive DNA

- Tandem Repeats - Interspersed Repeats

AR Diseases: most common

- Tend to be early and severe; death in childhood common - +/- lactic acidosis - Mostly brain, muscle - wheelchair, MR, seizures - Can involve other organs - Phenotypes not predict-able; if Complex subunits not involved, ETC function test may be non-diagnostic

Genetic defects of Hb (Incomplete Polypeptide)

- Thalassemias - Drastic truncation of polypeptide, therefore no or very limited function. - Cannot make the normal alpha/beta dimers. - Sometimes can activate fetal Hb subunit chain that can substitute (poorly) for an adult subunit.

ELASTIN

- The "rubber" protein forms a 3-dimensional network of cross-linked polypeptides. 1. Primary structure - mostly small nonpolar AAs, plus lot of Pro (causes kinks) and Lys (K) (sites for cross-linking). 2. Lysine crosslinks - 2 chains together with 4 cross-links - called desmosine (initiated by lysyl oxidase). 3. Reversible stretching. Elastin helps large arteries act as a secondary pump in circulatory system because heart is a pulsatile pump > stores energy & smooths out blood flow.

Fine-Tuning of O2 Delivery (O2 concentration - positive allosteric regulation) (How HB responds to differences in pO2 in lung & tissues)

- The 4 chains in deoxy form are held together in part by 8 ionic bonds; called T (taut or tense) form. - Upon oxygen binding to a heme: a structural change within a subunit (heme oxygenation) causes a structural change at the subunit interface.

Bottom Line

- The basis for enzyme-mediated rate enhancement. - How a pathway or tissue can be affected by a mutant or defective enzyme.

Globin proteins w/ heme complex

- The gas-binding site: heme prosthetic group is a complex of iron (Fe2++ - not Fe3+ cuz that's rust) and porphyrin (no amino acid can do the job). - hemes in the globin protein chains of Hb, Mb are shielded from oxidation by water (hidden inside protein pocket to avoid formation of nonfunctional methemoglobin (MHb).) - Angled bond with O2 (because of position of the distal His residue) prevents extremely strong binding of Fe2++ to endogenously produced carbon monoxide.

Bottom Line

- The general structural features of the three main fibrous proteins. - The biosynthesis and the assembly of the normal collagen fibril. - Fibrous protein defects caused by mutant genes or metabolic perturbations. - do not need to memorize specific collagen or disease types (III), specific mutations (E342) or codons, etc.

FACTOR XA PRODUCTION CASCADE OVERVIEW

- The initiation of the coagulation cascade is due to components of exposed /wounded subendothelial cells. - The final steps are on the lipid surface of activated platelets.

Bottomline

- The major structure/function relationships of hemoglobin. - The allosteric control mechanisms for Hb response to pH, CO2, and BPG. - The major problems caused by common Hb mutants or poisoning...and any cures. - do not need to memorize specific Hb mutations (E342) or codons, etc.

Mitochondrial genome: the other human genome

- The mitochondrial genome is composed of a single compact circular chromosome - The mitochondrial genome is maternally inherited - Heteroplasmy: Each cell may have different numbers of mutant and normal mitochondrial genomes

Bottom Line - KNOW:

- The nature of weak forces. - The four levels of protein structure. - The critical features of polypeptide structure and of bonding interactions that govern folding. - The concept of protein conformation. - The general basis of molecular disease (e.g., mutant gene => defective or missing protein => perturbed activity or lack of function).

G6PDH (cont'd) (STOP SIGN)

- The red blood cells of males and homozygous females with only the mutant enzyme are much more sensitive to oxidant damage induced by certain drugs (e.g., antimalarials and sulfa drugs) or certain foods (e.g., favism from broad beans). - Patients develop hemolytic anemia episodes about 2-3 days after exposure. * genes and environment are intertwined in health or disease!

Thrombin Amplification

- Thrombin can also activate some of the early parts of cascade by triggering zymogens and factors (Factor V, VIII) - for a burst of clotting - feedback amplification for even higher amounts of thrombin production.

clot formation

- Thrombin converts soluble fibrinogen into insoluble fibrin strands. - This clot is much more effective at stopping bleeding than the platelet plug alone.

Constitutively Nuclear

- Thyroid Receptor - Retinoic Acid Receptor - Retinoid X Receptor

Clot initiation

- Tissue factor is released from exposed cells and starts a coagulation cascade to turn on thrombin

Dysplasia

- abnormal, disorderly growth - often leads to cervical cancer in conjunction with chronic infection

Intrinsic pathway

- Triggered by RNA of the injured cells (contact phase). - Factor XII binds to this surface and becomes 105-fold more active. - Proteolytically clips more XII (autoactivates) and activates various complexes of [kallikrein, high molecular weight kininogen and XI] by cleavage. XIa then cleaves Factor IX. - IXa subsequently cleaves X; a cascade that makes Xa. - VIIIa is a helper factor that stimulates IXa 105 fold (deficient in hemophiliacs) but it is not a protease itself. - FACTOR XII BINDS TO RNA=>XIIa => activates XI to XIa=> XIa activates IX to IXa (gets helped by VIIIa)=> IXa activates X to Xa

Activation in the correct location:

- Trypsin needs to be active in the early part of the small intestine for effective digestion - Glucagon is required in the systemic circulation (regulation of glucose metabolism)

reticular

- Type III collagen - delicate supportive framework

Ubiquitination (STOP SIGN)

- Ubiquitin activating enzyme - E1 - Ubiquitin conjugating enzyme and ligase - E2/E3 - >300 distinct ubiquitin ligases, each targets a different kind of structurally aberrant protein or a normal short lived protein

Depends on sex of transmitting parent

- Unstable triplet repeats, typically >40 may expand in subsequent generations - Many disorders caused by triplet repeat expansions show anticipation - Expansions generally increase when inherited from either the mother or father - Unstable repeat lengths may contract slightly, but not significantly and not to the stable range

X Chromosome Inactivation Random (Text)

- Usually X-inactivation is random - Sometimes skewing of maternal / paternal X-inactivation - X-controlling element (Xce) alleles may affect choice; may cause skewing - Chromosome abnormalities can affect the randomness of X-inactivation - A structurally abnormal X is preferentially inactivated (e.g. large deletion of the X-chromosome) - The normal X is inactivated in the case of X-autosome translocation

Symptoms/Diagnosis of Stroke

- Usually significant neurologic deficit although symptoms may be subtle - In a young child refusal to walk may be a symptom of pain or stroke - MRI is the imaging study of choice - These patients should be transferred to a major medical center

Factors affecting the manifestation of mitochondrial mutations

- Variable number of mitochondria per cell (2-100) and mtDNA chromosomes per mitochondrion (5-10) - Heteroplasmy: Different cells / tissues may have varying numbers of mutant mtDNA - Variable susceptibility of different tissues to defective oxidative phosphorylation caused by abnormal mitochondrial function

Scurvy

- Vitamin C is required for hydroxyproline formation - Deficiency leads to a triple helix that is not stabilized by extra H-bond from this special AA, - Therefore triple helix "melts" at body temperature.

Oxidation of heme

- When His that binds Fe atom is changed, a H2O molecule is allowed to oxidize Fe > brown arterial blood, blue-ish skin. - This problem leads to cyanosis (blue disease) and methemoglobinemia (where higher concentrations of methemoglobin which has lower affinity for O2 & can be made worse if G6PD issues, nitrates).

Recognized as malformation syndromebefore the genetic cause was known

- Williams syndrome - Velocardiofacial syndrome (VCFS) AKA DiGeorge or Sphrintzen - Langer-Giedion syndrome - Prader-Willi - others

Sickle Cell Anemia A -

- a "sticky" spot that allows DEOXY form of HbS to polymerize distorting red blood cell shape from normal round shape into a crescent (sickle) shape that clogs up capillaries especially in regions of low oxygen tension. Prevention: Avoid getting out of breath & stay hydrated!! More later at the Clinical Correlation

allosterism

- a change in activity induced from a site other than the active site

Isochromosome

- a chromosome that has lost one of its arms and replaced it with an exact copy of the other arm.[1] - This is sometimes seen in some females with Turner syndrome or in tumor cells. - This may also cause an isochromosome to have two centromeres

Multifactorial Inheritance

- a combination of genes and environment (multiple sclerosis) - Recurrence estimates depend on empiric data 3-5% for 1º relative increases with more affected family members decreases for more distant relatives higher when severity of index case is high consanguinity increases recurrence Multifactorial disorders include: Isolated cleft lip NTD Pyloric stenosis

Tay Sachs

- a common lipid storage disease that results from the complete loss of a single enzyme, hexosaminidase A, therefore lipids accumulate in cells causing severe and ultimately fatal disease by age 3. - If enzyme levels are 10-20% of normal, then lipid accumulation does not cause problems until midlife.

Incontinentia Pigmenti

- a genetic disorder that affects the skin, hair, teeth, nails, and central nervous system. It is named due to its microscopic appearance. - X-linked Dominant

RNA-induced silencing complex (RISC) in translation supression

- a multiprotein complex that incorporates one strand of a small interfering RNA (siRNA) or microRNA (miRNA) - the DICER cleaves pre-miRNA or pre-siRNA - uses the siRNA or miRNA as a template for recognizing complementary mRNA. - When it finds a complementary strand, it activates RNase and cleaves the mRNA.

Alzheimer's Disease, Creutzfeldt-Jacob Disease, Mad Cow Disease, and Scrapie (STOP SIGN)

- all result from conformational change in normal soluble protein found in brain - The affected protein adopts a conformation that is insoluble and precipitates. - And to make matters worse, other "good" protein molecules will also become insoluble when they contact the precipitate. - These precipitates (which are not readily degraded by proteases or lyosomes) steadily grow in the brain over time leading to neural tangles that ultimately result in necrosis of brain tissues. - The loss of brain tissue (makes a spongiform appearance) cause the symptoms of memory loss in the elderly or infected.

Pseudostratified columnar

- all the cells touch the basal membrane - some touch surface but not all do - not truly stratified (cells are parallel but do not reach surface)

Technology

- allows quicker, cheaper genome sequencing - already in use - will get to $1000 per human genome - Whole genome sequencing of cancers will become standard of care (short reads can be assembled into a genomic sequence) - needs to deal with Intratumoral heterogeneity which may thwart targeted therapeutics (due to accumulating mutations lead to multiple malignant clones which allows tumors to become more adaptive)

Sticky spot of sickle cell anemia

- allows the deoxy form of HbS to polymerize distorting the red blood cell shape from the normal round shape into a crescent (sickle) shape that clogs up capillaries especially in regions of low oxygen tension.

MSI reflects decreased DNA repair

- appearance of novel STR (short tandem repeats,a.k.a, microsatellites) alleles - caused by defective mismatch repair - in colon cancer:better prognosis

Systems medicine

- application of systems biology to health - Blood:"diagnostic window for disease analysis" - "Multiparameter" testing: 2,500 markers measured & analyzed simultaneously - Reactive vs.preventive medicine - Knowledge of current state of system allows prediction of future state ('emergent properties' of system) - "determination of a probabilistic future health history for each individual"

Poisoning by Methanol (wood alcohol) or Ethylene Glycol (automobile antifreeze)

- are actually caused by the conversion of the initial compound into toxic metabolites (formaldehyde, formic acid, and oxalic acid) by alcohol dehydrogenase in the body.

segmental duplication

- are mutational hotspots for large deletions/duplications - tagged along with unequal recombination

Subendothelial cells

- are normally not exposed to blood until injury. When exposed by injury, platelets adhere to subendothelial cells.

platelet cells

- are small vestigial cells formed in healthy (!) bone marrow. - normally circulate in an inactive form. - if encounter subendothelial cells, then bind & activate.

Clotting factors

- are soluble plasma proteins made in healthy (!) liver that circulate in inactive form. - Cascades on activated platelet surface turns on thrombin, a serine protease, that converts soluble fibrinogen into insoluble fibrin that glues platelets and wound together.

Triplet repeat sequences: (questions)

- are stable when in number

stratified Squamos non-keratinized

- basal cells are more of a cuboidal shape - but by the time we reach the surface, the surface layer is simple squamos

granulocyte

- basophil - neutrophil - eosinophil

exanguination

- bleeding to death

Hematoxylin

- blue - basic dye - positively charged - binds to the negatively charged acidic substance (that is basophillic) - nucleic acids (phosphate), & cartilage matrix (sulfate groups)

osteoclasts

- bone resorbing cells - Large multinucleated cells. - Derived from fusion of monocytes. - Function is bone resorption. - Acidify matrix and release hydrolytic enzymes onto bone surface.

Osteogenesis imperfecta

- bone-brittle disease - Typically a point mutation (usually glycine to X) impairs triple helix formation. - Particularly bad for the structure when mutation at the C-terminus (collagen zips up C=>N direction) and the folding process stalls out early.

Poisoning (CO-carbon monoxide)

- breaking down heme generates CO so ~1% CO:Hb in blood naturally (lucky for the angled heme/gas bond). - CO binds Hb 200X more strongly than O2 thus low [CO] can kill over time. - Brain and heart are most susceptible to hypoxia so most deaths secondary to dysrhythmias and infarction. - CO concentrated by fetus (fetal Hb has higher CO affinity than adult Hb)=> low birth weight, wasting, & neurologic deficit. - Hazards: bad home furnace, leaky car exhaust system, methylene Cl-based paint stripper (liver converts into CO) or smoking

mRNA of Tumors

- can be extracted from samples and quantified using cDNA & probe hybridization - relative expression of genes can yield a distinctive signature (like a fingerprint)

Ethanol (as inhibitor)

- can be used as a competitive inhibitor to keep the methanol or ethylene glycol from the active site. - Therefore, the patient is kept drunk (high ethanol concentration!) so that the toxic metabolites are not formed and the poison is excreted unchanged.

Question: Genomic disorders

- can encompass single gene disorders

Emerging hallmark :cancer cells escape detection

- cancer cells can turn off antigen expression and secrete antiproliferative factors

Different mechanisms of cancer

- cancer growth disrupts adjacent structures (organ obstruction) - metastatic disease (common site-liver) - can invade blood vessels and lymphatics - can invade nerve cells - can metastasize through peritoneal cavity - benign tumors can kill if in critical location

Beta Bend/Turns

- carbonyl of n residue H-bonds with amine of n+3. - This structure is useful for reversing direction of chain near protein surface to get chain back inside. - Common residues in turns: glycine good since no side group to cramp; proline starts a kink for a tight turn.

Specialized Connective Tissue

- cartilage - bone - adipose - blood - hematopoeitic insert slide 19

Burkitt lymphoma

- caused by Epstein-Barr Virus (EBV)

Body cavity lymphoma & Kaposi sarcoma

- caused by human herpes virus 8 (HHV-8)

Cervical Carcinoma

- caused by human papilloma virus (HPV)

sunlight (UV light)

- causes carcinoma, melanoma - through pyrimidine dimers

aflatoxin (from grain contaminated with Aspergillus)

- causes hepatocellular carcinoma - through reactive metabolite causing DNA damage

radiation (ionizing)

- causes leukemia,thyroid cancer - through mutations, genome-level breakage

cigarette smoke (3, 4 benzyprene)

- causes lung cancer - through reactive metabolite causing DNA damage

asbestos

- causes mesothelioma - mechanism no one knows

Hypercalcemia

- causes squamous cell lung cancer; others - uses parathyroid hormone-like products

Angle around peptide bond

- chains rotate around alpha carbon - but peptide bond stays the same

OVERVIEW OF AMINO ACIDS

- chiral L-amino acids with same basic structure centered on the alpha-carbon. - amino group (+NH3): positive charge at pH 7.4. - carboxylic acid group (COO-): negative charge at pH 7.4. - R-group : side chain or substituent - variable properties

Velocardiofacial syndrome

- chromosome 22q- - Learning disabilities - IQ 70 - 90 on average - Cleft palate - Velopharyngeal incompetence - Tapering fingers - Congenital heart defects - Renal anomalies - seizures

tertiary

- clusters of local structures - interactions and optimal packing of motifs and a hydrophobic core

4. Some use cofactors

- cofactors have special chemical properties not available to amino acids to alter substrate a. Metal ions (e.g. Fe2+, Zn2+, Mn2+) accept electrons, or engage in reversible bonding with ligands. About 2/3 of enzymes need a metal group. b. coenzymes - diffusible (e.g. the NAD, FAD groups donate/accept H- or 2H). c. prosthetic groups - covalently bound (e.g. biotin group is a CO2 carrier).

Collagen Role - CELL ADHESION

- collagen fibrils interact with other cell surface and matrix molecules (e.g., integrins, fibronectin, laminin & proteoglycans) which in turn bind to other cell surfaces.

Motifs

- combinations of helix, sheet, etc. structures - "Diagnostic signature" for certain functions or types of proteins (e.g., helix-turn-helix motif of DNA-binding proteins)

connective tissue

- consists of cells dispersed in extracellular matrix - mostly matrix w/ relatively few cells - categorized based on morphology

Conformational change (fluctuation) from one state to another

- control activity (e.g., protein off or on depending on a phosphorylation event that changes tertiary structure). - fine-tune function with feedback (e.g., do not synthesize too much product if not required). - accommodate several types of molecules (e.g., an enzyme can accept a substrate and release a product). - convey information (e.g., hormone in blood binds to a cell surface receptor that starts a signaling cascade in the cell cytoplasm).

Y-linked

- could be linked to decreased fertility and ear hair

How are amino acids linked?

- dehydration reaction - The formation of the amide or peptide bond occurs in the ribosome. - The loss of a water molecule leaves an amino acid "residue". - Proteins are directional: "N" and "C" termini. - Peptide bond can H-bond with other peptide bonds & groups (prevalent!).

- gen ending

- denotes a zymogen

Kabuki Syndrome

- developmental delay, distinctive facial features - exome sequencing id's MLL2 mutations as cause of disease

juxtacrine

- direct contact (cell-cell contact; synapse; gap junction)

Post-translational modifications also require

- disulfide crosslinking - two cysteine residues make a S-S bond - phosphorylation of S,T,Y (make protein turn on or off) - glycosylation of S,T,N (for recognition and/or increase solubility) - proteolytic processing (zymogens, preproproteins; how to tame a vicious digestive enzyme until needed) - accessory/co-factors (heme for oxygen binding) since AAs cannot do all types of chemistry

zymogen

- dormant form of protein

When organized properly, a combination of all these forces can:

- drive the initial folding of a protein structure (e.g., hydrophobic core). - allow the protein structure to hold together noncovalently using weak (1-5 kcal/mol) bonds into a precise structure. - allow the protein to attract/bind/alter its substrates (ex. substrate is hydrophilic and negative, so perhaps the active site of the protein is hydrophilic and positive).

Components

- endothelial cells - subendothelial cells - platelet cells - clotting factors

Four basic tissue types

- epithelial - connective - muscular - nervous

Triplet Repeat Expansions - Gain of Function

- expansions usually <100 - within exon - codes for polyglutamine tracts

Cystic Fibrosis

- fatigue - chronic cough - recurrent URIs - thick, sticky mucus - chronic hypoxia - decreased absorption of vitamins & enzymes - abdominal distention - decrease digestive enzymes - rectal prolaspe (fatty, stinky stools) - meconium ileus in Newborn - in males (CBAVD)

FISH

- fluorescense in situ hybridization - best way to diagnose chromosome microdeletions - detection of aneuploidies - identification of chromosome translocation - analysis of chroms by hybridizing a fluorescently labeled probe to chroms -hybridize metaphase chroms (dividing cells) -hybridize interphase nuclie of non-dividing cells -chromsome painting - allows for rapid diagnosis of prenatal chromosomal aneuploidies

megakaryote

- found in bone marrow - platelet forming cells

Transitional epithelium

- found in urinary bladder - specialized for distention - protein plaques at the apical side of the lumen

Multilocular fat cells (brown adipose

- generates heat in newborns - Heat is generated by the uncoupling of ATP synthesis to the H+ gradient produced by mitochondrial enzymes (electron transport chain).

Mitochondrial disorders

- genetic defect involving mitochondrial DNA (maternally inherited) - Rare - e.g. Laber hereditary optic neuropathy (LHON)

CpG islands

- genomic regions that contain a high frequency of CpG sites.

secondary

- helix, sheets, turns, etc. - bonding networks at short range (a few AAs up & down chain, OR close in 3-dimensional space).

N-linked glycosylation, O-linked glycosylation

- help with trafficking to appropriate location

General Mechanism of H&E staining

- hematoxylin - eosin

Proteins as Drugs

- hemophiliacs need the clotting protein Factor VIII. - SCID sufferers (severe combined immunodeficiency) require injections of adenosine deaminase. - diabetics use insulin to control blood sugar levels. - dwarfism can sometimes be circumvented with growth factor. - heart attack victims benefit from quick administration of clot-busters like streptokinase or tissue plasminogen activator (t-PA).

Hepatocellular carcinoma

- hepatitis B virus (HBV) & hepatitis C virus (HCV)

Clot-busters or Thrombolytics

- human proteins tPA or urokinase, or the bacterial protein streptokinase that activate plasminogen to plasmin. - given through I.V. within 1st hour of a heart attack or stroke to limit size of infarction. (tPA should be better since it will localize to the clot). - reduction in clot size (or hopefully elimination) allows more blood to get to the oxygen-starved cardiac or brain tissue and limit the size of the necrotic zone. - But need to monitor patient (using lab tests such as PT, PTT, fibrinogen; more focus in Spring course) and have platelets and Factor VIII on hand.

3 types of Cartilage

- hyaline - elastic - fibrocartilage

Nuclear Hormones

- hydrophobic (membrane permeable) - Bound to carrier proteins in blood - long persistence in blood (h to d)

Enabling characteristic

- inflammatory cells can promote cancer

Aspirin

- inhibits cyclooxygenases irreversibly. - These enzymes make two small molecules involved in clotting process. a) Platelets make thromboxane A2 to recruit other platelets. b) Endothelial cells make prostaglandin I2 that inhibits platelets outside of clot. - Since platelets are "dead", they cannot regenerate their supply of cyclooxygenase, but the endothelial cells can make more enzyme. - Therefore aspirin will inhibit clotting, but not perfect since endothelial cells somewhat affected, too.

Chorea

- involuntary, non-repetitive, jerky movements

Mast Cell

- involve in allergic reactions

Small nuclear ribonucleoprotein-associated protein N

- involved in mRNA splicing - The small nucleolar RNA (snoRNA) HBII-52 has been localized to the Prader-Willi critical region and is thought to be important in processing an mRNA expressed from a gene located on a different chromosome.

2. Vast majority of interactions between substrate and amino acids' active site are NONCOVALENT

- ionic bond, hydrogen bond, van der Walls, and hydrophobic interactions a. These forces may guide substrate into cleft or pocket of active site (where reactions will not have to compete with bulk solvent water). b. The shape and polarity of substrate may match the active site (lock-and-key model of complementarity).

Disseminated Intravascular Coagulation

- is a pathological activation of coagulation (blood clotting) mechanisms that happens in response to a variety of diseases. - leads to formation of small blood clots inside blood vessels throughout body - As the small clots consume coagulation proteins and platelets, normal coagulation is disrupted and abnormal bleeding occurs from the skin, GI tract, respiratory tract and surgical wounds. - small clots also disrupt normal blood flow to organs (such as the kidneys), which may malfunction as a result.[2]

Triplet repeats

- islands in the genome containing three base pairs repeated over and over - The normal number of repeats is generally less than 40

primary

- just the amino acid sequence

DNA replication is semi-discontinuous

- lagging strand - Okazaki fragments stuck together by ligases

Endothelial cels

- line the interior of blood vessels; when these cells are intact, inhibits coagulation, enhances clot digestion, and inactivates platelets.

Mesothelium

- lines body cavities and covers free surface of organs. - simple squamos epithelial;

Endothelium

- lines the lumen of blood and lymphatic vessels - classified as simple squamos endtoelim

epithelial

- lines tubes and covers surfaces (intestine, or skin) - cellular - comprised of sheets of cells - categorized based on morphology

Kinetochore

- located at the centromere and is essential for accurate chromosomal segregation during cell division

Example: Inbred family: 3 affected children (2 sibs)

- look for shared regions of homozygosity among the offspring - Homozygosity mapping allowed zooming in on (large) region of interest: 10cM/14Mb and containing 85 annotated genes. - KCTD7 was strongly considered after bioinformatics analysis of the region because potential implication in K+ conductance and because of the known pathogenic role of potassium channel subunit mutations in some rare forms of epilepsy

STOP trial

- looked at: Whether TCD would predict children at risk of first stroke Whether transfusion therapy would be effective in primary prevention of stroke - subjects: 130 children w/ sickle-cell SS

Agranulocytes

- lymphocyte & monocyte

collagen

- made by fibroblasts - type I most abundant

bone organs

- made up of bone tissue - other CT, fat cartilage, bone marrow, blood vessels, and nerves

scurvy

- manifestaion of a vitamin C deficiency

Ehlers-Danlos Syndome

- manifestation of genetic defects

Southern Blot

- method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. - Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. - The method is named after its inventor, the British biologist Edwin Southern.

Serosa

- mosotlium + underlying connnective tissue

stratified cuboidal

- mostly in glandular ducts

Mitochondrial disorders

- muscle weakness or exercise intolerance - heart failure or rhythm disturbances - dementia - movement disorders - stroke-like episodes - deafness - blindness - droopy eyelids - limited mobility of the eyes - vomiting - seizures

stratified epithelium

- named for the cell shape that forms the most superficial layer - layers on top of one another - squamos (non-keratizined) - cuboidal - keratinized - columnar

Three-dimensional assembly of a single polypeptide chain.

- native conformation or structure folded "correctly" mostly by weak bonds; tightly packed interior (van der Waals). - Some proteins can have more than one conformational state, too - disulfide linkages help hold shape together - domains: independent folding units that have own function and used for mutliple purposes; ex - exons encoding a domain that are duplicated and swapped..

Heparin

- negatively charged polysaccharide helper factor gets ATIII and thrombin together. - a potent and widely used anticoagulant. Usually injected (charged molecule not absorbed in intact form by gut). - may be used to combat disseminated intravascular coagulation [DIC] that results from infection, drug complications, or other acquired disease. - If need to reverse effect, the antidote is protamine sulfate, a positively charged small protein that complexes with heparin. (note: low MW heparin has other characteristics)

Synapse

- neuron-neuron - neuron-muscle neurotransmitter release through the synaptic cleft between 2 neurons

Granulocyte differentiation

- neutrophilic myelocyte (last stage capable of mitosis) => neutrophilic metamyelocyte => neutrophilic band cell

Isoelectric point [pI]

- no net charge; pKa values averaged. - The ionization state of critical groups will determine if protein is functional.

Juxtracrine signaling

- notch pathway - synapse - gap junction

Hayflick limit

- number of times a normal human cell population will divide until cell division stops. - once senescence is reached, further cell division stops (little or no telemorase activity) - cancer cells do not have such limit (due to high telemorase activity)

HER2

- overstimulation of growth factor receptor - can immunostain for HER2 and also use FISH to look for amplification (HER2/centromere>2.2)

Thrombin Production Cascade

- overview: A series of amplification steps, a cascade, yields a large amount of active thrombin protease (Factor IIa) from inactive prothrombin zymogen (Factor II). - control: Regulatory helper molecules (positive effect) and protease inhibitors (negative effect) help to localize clotting to wound site (don't want all the plasma proteins throughout bloodstream coagulating!)

metacentric

- p & q are of equal length;

acrocentric

- p arm is so short, but still exists

Mutated gene for Li Fraumeni Syndrome

- p53

Quaternary

- packing of proteins next to other proteins.

A single amino acid change can lead to disease.

- perturb the structure/conformation of a protein molecule, and thereby alter its function [ex. collagens in osteogenesis imperfecta]. - remove a critical residue resulting in an inactive or nonfunctional protein [ex. Factor VIII in severe hemophilia A]. - alter the interaction (either more or less) of one protein with another protein [ex. sickle cell hemoglobin in hemolytic anemia]. - destabilize a protein resulting in a shorter biological half-life, thus leading to deficiency [ex. glucose 6-P dehydrogenase variants in GP6-DH deficiency (drug sensitivity and favism)].

Addition of small chemical groups

- phosphorylation

pKA

- point at which 50% of each form is present - Titration curve, buffer-like behavior; at the pKa = 50% in each form. Dissociation depends on environmental pH: @ 1 pH unit off pKa value, 90%/10% ratio. @ 2 pH units of pKa value, 99%/1% ratio. @ 3 pH units of pKa value; 99.99%/.11 ratio

Tandem Repeats may be polymorphic

- polymorphic microsatelittle repeats - DNA polymorphism: If a change in the genetic sequence is present at a frequency of at least 1% in the population

cervical intraepithelial neoplasia (CIN)

- potentially premalignant transformation and abnormal growth (dysplasia) of squamous cells on the surface of the cervix - is not cancer, and is usually curable - small % of cases can lead to cervical cancer

P4 of medicine

- predictive - preventive - personalized - participatory

premature stop codons

- premature STOP codons induce nonsense mediated RNA decay -EJC (exon junction complex) act as a memory of exon-exon junctions -EJCs are removed by 1st ribosome during translation

Vitamin K antagonists (coumarins-coumadin & warfarin)

- prevent gamma-carboxyglutamate formation by allowing vitamin K depletion, which causes clotting factors not to localize on the activated platelet surface. - are slow acting since the clotting factor half-life is 1 to 5 days; all of the factors made prior to coumarin treatment must be eliminated before the newly synthesized clotting factors w/o the carboxyglutamate modification predominate (but do not want to wipe out all modification!). - must monitor blood levels of drugs - if overdose, then inject vitamin K (since drugs are competitive inhibitors) - watch out for dehydration (drug levels increase as you lose water)

Proper protein structure is required for function.

- primary structure - secondary structure - tertiary structure - quaternary structure

Autocrine signaling

- prostaglandin & interleukins acting on originating cell

Secretory Route

- protein undergoes many steps as it is pulled along from the inside to the outside of cell

What holds a protein together? What makes a protein work?

- proteins are molecules with delicate structures that are forged from WEAK bond interactions of amino acids with each other and their substrates. - Proteins fold into particular "shapes" or structures as guided by forces: IONIC BOND (AKA SALT LINKS) HYDROGEN BOND HYDROPHOBIC FORCE/INTERACTION van der WALLS FORCE

3 Types of Post-translation modifications

- proteolytic cleavage - chemical modification - folding

submetacentric

- q is longer than p;

Systems biology

- quantify systems components and group into networks

Eosin

- red - acidic dye - negatively charged - binds to positively charged basic substance (that is acidophilic) - protein (amino groups)

Specialized connective tissue: Blood

- red and white blood cells suspended in plasma - agranulocytes & granulocytes

nonsense mediated decay

- reduces errors in gene expression by eliminating mRNA transcripts that contain premature stop codons

Paternal Isodisomy (UPD 14)

- result of trisomy rescue - polyhydramnios, thoracic and abdominal wall defects, growth retardation, severe developmental delay and dysmorphic facies - paternally expressed genes DLK1 and RTL1

Maternal Isodisomy (UPD 14)

- result of trisomy rescue - pre and postnatal growth retardation, hypotonia, joint laxity, motor delay, precious puberty, and minor dysmorphic features of the face, hands, and feet - maternally expressed - several (sno)RNAs and microRNAs, GTL2, RTL1as, and MEG8

Sickle cell anemia A

- results from a defect in the hemoglobin [Hb] beta subunit chain in which glutamic acid #6 has been converted to a valine (HbS) as a result of an A to T point mutation. - This forms a "sticky" spot that allows the deoxy form of HbS to polymerize distorting the red blood cell shape. - Heterozygous patients (1 in 12 occurrence in blacks) with 50% normal Hb, 50% mutant HbS have mild form of disease. - Homozygous patients (1 in 500 occurrence in blacks) are very sick since all the beta chains are mutant HbS.

Osteogenesis imperfecta

- results from a point mutation that changes glycine to a bulky amino acid that impairs triple helix formation. - Strong collagen fibers do not form resulting in "brittle bone disease" (1 in 15,000 occurrence). - can be lethal (due to bad bones)

Homozygosity mapping - concept

- revolutionize ability to track homozygosity much better - offspring in box has (1/4) of being affected with mutation

Ehlers-Danlos syndrome

- rubber or elastic man - Heterogeneous group with: stretchy skin, loose joints, poor wound healing, failure of vessel/organ structures. - Typically caused by no cross-linking (no strengthening) OR Failure to remove N-peptide (collagen not converted to most insoluble form).

Paracrine signaling

- secrete hormones or gfs that act on adjacent cell ex - glucoagon & somatostatin acting on insulin production

Endocrine signaling

- secrete polypeptide or sterioids (into blood vessel) ex - thryotrophic hormone, estradiol

Osteogenesis Imperfecta

- severe, lethal type of manifestation of genetic defects

Human papillomavirus (HPV) causes cervical cancer

- sexually transmitted - dsDNA virus - 40 types (out of 100) infect female GU tract - only minority of infected women develop cervical cancer - screening for cervical dysplasia (pap smear & molecular tests)

autocrine

- signal is excreted and targets same cell

intracrine

- signal produced w/n targeted cell

MicroRNAs regulate gene expression

- size and shape:21-23 bases;stem-loops or linear - functions:noncoding;downregulate gene expression in development,oncogenesis - expression:tissue-specific - number:10,000s? (c.f.,approx.20,000 protein-coding genes) - location:introns,exons,elsewhere - organisms:plants and animals - can have tissue-specific expression

Unilocular fat cells (white adipose)

- small lipid droplets in cytoplasm fuse to form a large droplet - lipid storage and mobilization

lacunae

- space occupied by cells

muscular

- specialized for contraction to produce movement - categorized by function

nervous tissue

- specialized to respond to stimuli and conduct signals from one point to another - categorized by function

osteoprogenitor cells

- stem cells of periosteum and marrow cavity

3 Types of Epithelial

- surface - glandular (endocrine and exocrine) - special (sensory and reproductive

Phenylketonuria (PKU)

- the most important inborn error of amino acid metabolism. - Phenylalanine hydroxylase is absent so phenylalanine [Phe] accumulates in the blood at 20- to 100-fold higher values than normal IF do not watch diet. - Somehow high levels of Phe & its metabolites causes mental retardation in infants.

columnar

- the taller the cell, the more absorptive, and vice versa - Goblet cell - unicellular exocrine gland that are in parallel with simple columnar epithelial cells

infarction

- tissue death (necrosis) caused by a local lack of oxygen, due to an obstruction of the tissue's blood supply.

CF & WBCs

- to fight infection, WBCs arrive at airway & release toxic molecules to kill bacteria - toxic molecules damage both bacteria, WBC & normal body tissue in airway - WBC DNA is released into the mucus (making it thicker) - Airways are damaged, leading to larger, dilated airways => bronchiectasis

nullisomy

- type of anueploidy - missing a pair of homologs - lethal preimplantation

3. Conformational shape change during catalysis

- use induced fit model to bring functional groups into play on the substrate. - After substrate is converted into product, the enzyme no longer "holds" onto the product and it diffuses away

hematemesis

- vomiting blood

Positional cloning of a disease gene after linkage mapping between closely linked polymorphic markers

- was very frustating - was how many genes were first identified

Squamos keratinized

- will have several layers of dead cells

Severe hemophilia A

- x - Linked - the result when one of the arginines at positions 1689 or 372 of Factor VIII is mutated to another residue that cannot be clipped by the protease thrombin; - therefore VIII is never transformed into the useful active form (1 in 10,000 occurrence in males). - if extrinsic had flaw, then no birth would even take place

PCR & Southern Blot

---detects large (>1kb) mutations -cut with restriction enzyme -gel electrophoresis -blotting to membrane -hybridize with specific probe PROCESS: -fragment DNA -run on gel -blot to membrane -hybridize with specific probe -uses X-ray film

Hemidesmosome

-Attachment to basal lamina -Bound by Integrin -

Plasma membrane

-Barrier between cytoplasm and extracellular environment -Composed of lipids, proteins, and carbohydrates -continuous double layer of lipids

Exceptions for "-omas"

-Blastoma (blast cells) -Glioma (glial cells, brain) -Lymphoma (lymphoid cells) -Melanoma (melanocytes) -Mesothelioma (mesothelium, lining of abdominal, pleural, and pericardial cavities) -Seminoma (germ cells, testicular) -Choristoma

cAMP Signalling Pathway

-But: Galphai proteins can inhibit adenylyl cyclase!

Membrane proteins

-Can be integral, surface, or integral -Can link the membrane to the cytoskeleton and extracellular matrix

Golgi apparatus

-Site of protein post-translational modification and "shipping" -cis (entry face) -trans (exit face) -transport to and from Golgi by transport vesicles

Nucleolus

-Site of ribosomal RNA synthesis -Contains loops of DNA encoding rRNA genes

Stereocilia

-Ultrastructurally identical to microvilli -Absorptive function (e.g. epididymis of male reproductive tract)

Constitutive Secretory pathway

-Default -Occurs in all cells -Continuous secretion of soluble proteins into the extracellular space

Microfilaments

-Double-Stranded helical polymers of actin -involved in cell movements -Highly concentrated beneath the plasma membrane -Actin -Associates with myosin to form contractile structures

Gap Junction

-Enables rapid flow of small molecules and ions between cells -Results in electrical and metabolic coupling between neighboring cells -Coordinates activities among cell populations -Formed by hexagonal arrays of connexins (called connexons) -Especially prominent in epithelium, smooth and cardiac muscle, nerve cells, and osteocytes -opening and closing regulated by calcium and phosphorylation of connexins

Polycythemia

-High hemoglobin - causes hepatocellular carcinoma, renal cell carcinoma, others - uses erthropoietin

Heterchromatin

-Highly condensed, inactive form of DNA -Methylated -excluded from region around the nuclear pores

Components of Cytoskeleton

-Intermediate filaments -Microtubules -Microfilaments

LOD SCORE ANALYSIS Determining the significance of linkage results

-JUST REMEMBER >ABOVE 3 = EVIDENCE OF LINKAGE & <3 = EVIDENCE AGAINST LINKAGE OR INCONCLUSIVE

Euchromatin

-Less-condensed chromatin -Actively transcribed

Microtubules

-Long, rigid, hollow cylinders made of protein tubulin -Typically have one end attached to "microtubule organizing center" (centrosome) -Polymerization and depolymerization of tubulin controls movement of transport vesicles and other organelles

Endocytosis

-Macromolecules in the extracellular fluid bind to cell-surface receptors -Clathrin binds to cytosolic side of receptor -Clathrin-coated vesicle taken in, quickly sheds to fuse with early endosome

Peroxisome

-Membrane-bound organelles specialized for carrying out oxidative reactions -Oxidase--use O2 to produce H2O2 -Catalase--H2O2 to H2O Responsible for detox!!

Lysosome

-Membrane-bound vesicles containing hydrolytic enzymes -Controlled digestion of macromolecules -pH 5, maintain through H+ pump

Microvilli

-Non-motile -Shorter than cilia (1 micron in length) -Absorptive function -Comprised of a core of actin microfilaments

Key microscopic features of malignancy

-Nuclear irregularities (atypia) --Irregular contours (jagged, notched) --Increased size (n/c ratio) --large and/or multiple nucleoli --"open" chromatin -Overall pattern --invade surrounding tissue --irregular 3-D

Nuclear pores

-Pore transport controlled by Nuclear pore complex -cytosolic nuclear import receptors bind to prospective nuclear protein (requires nuclear localization signal) -Complex guided to pore by fibrils -Binding of nuclear protein opens pore -Complex transported into nucleus -receptor back to cytosol

Regulated secretory pathway

-Present in cells specialized for secreting products in response to extracellular signals -stored in high concentrations in secretory vesicles -Release after specific stimulus

Rough Endoplasmic Reticulum

-Ribosomes bound to membrane surface for the synthesis of transmembrane proteins and lipids -import of proteins occurs co-translationally -all proteins destined for secretion synthesized in rough ER

Cachexia

-Wasting -Causes many cancer types - uses Tumor necrosis factor

MLPA (Multiplex Ligation-dependent Probe Amplification)

-can detect exon abnormalities ex: MLPA can detect exon deletions or duplications in the dystrophin gene.

Nuclear Envelope

-composed of 2 phospholipid bilayers -continuous with endoplasmic reticulum -Perinuclear space between membranes -Membranes fuse in places to form nuclear pores

DNA Cloning Steps

-cut out DNA of interest with restriction enzyme -insert into vector (virus) -transfer into bacteria -replication in host cell -antibiotic selection of bacteria containing the desired plasmid -isolate plasmids and cut out of vectors with restriction enzyme =Multiple, identical copies of the inserted DNA of interest

Emerging hallmarks of cancer

-deregulating cellular energetics -avoiding immune destruction

Features of benign tumors

-encapsulated -small, regular nuclei -inconspicuous nucleoli -low N:C ratio -not mitotically active -no necrosis -not metastatic

Mitochondria

-generates ATP used to drive cellular reactions -single circular mDNA -import O2, ADP, and NADH (from citric acid cycle) and exports CO2 and ATP -protein import required molecular chaperones

Enabling characteristics of cancer

-genome instability and mutation -tumor-promoting inflammation

Polymerase Chain Reaction

-in vitro method for amplifying DNA -millionfold amplification in ~2hrs -PREFERRED METHOD FOR AMPLIFYING DNA (replaced cloning) advantages: -identifies minute quantities of DNA -can detect specific DNA sequences -mutation detection -DNA sequencing -polymorphism detection -DNA fingerprinting -diagnosis of infectious diseases (TB, HIV)

Features of malignant tumors

-infiltrative -pleomorphic nuclei -prominent and irregular nucleoli -high N:C ratio -mitotically active w/ abnormal mitotic figures -areas of necrosis (lack blood suppy) -metastatic -jumbled together (don't respect their neighbors)

CpG dinucleotide

-most common site for mutation causes -spontaneous deamination of methylated cytosines -mutation hotspot with 8.5 timex greater mutation rate over other dinucleotides -not all CpG duos have the methyl group on Cytosine (making it 5-methylcytosine) ****IF YOU deaminate cytosine= no biggie; efficiently recognized and repaired, you get uracil ****IF YOU deaminate 5-methylcytosine = TROUBLE; not recognized, you get thymine

dot blot

-not as easy as PCR and sequencing ex: can detect sickle cell disease

Chronic myelogenous leukemia

-presentation: anemia/fatigue -t(9;22) balanced translocation or BCR-ABL required for diagnosis & can be IDed w/ cytogenetics -Targeted molecular therapy available (imatinib) - spectrum of malignant granulocyte cells in the peripheral blood - can be mimicked by increase in WBCs due to infection

Hallmarks of Cancer

-resisting cell death -sustaining proliferative signaling -evading growth suppressors -inducing angiogenesis -enabling replicative immortality -activating invasion and metasasis

DNA Sequencing (Sanger & "nextgen")

-the process of determining the precise order of nucleotides within a DNA molecule. - It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA. - The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. - OUHSC is getting a new next generation sequencer.

Sldie 22

...

cuboidal

...

Familial mutation of Rb

1 mutation is already passed down, so must only have 1 random mutation to develop retinoblastoma

Cystic Fibrosis: Pathogenesis

1) Increased mucus viscosity with obstruction lungs, pancreas, GI tract, liver, gall bladder, reproductive tract 2) Respiratory tract colonization/infection with "unique" organisms 3) Increased sodium & chloride in serous secretions

Medical Management

1. Clot-busters or Thrombolytics 2. Heparin (anticogulant) 3. Vitamin K antagonists (coumarin) 4. Aspirin 5. E-aminocaproic acid (EACA) and transeamic Acid

Protein Kinase A (PKA)

1. Cytosolic cAMP increases 2. Two cAMP molecules bind to each PKA regulatory subunit 3. The regulatory subunits move out of the active sites of the catalytic subunits and the R2C2 complex dissociates 4. The free catalytic subunits interact with proteins to phosphorylate Ser or Thr residues

platelet activation

1. Fibrinogen receptor appears on platelet surface a. Fibrinogen helps glue the platelets together (platelet aggregation) to form a platelet plug (within a few minutes). b. This plug can slow bleeding but fragile and it may re-bleed. 2. Also phosphatidyl serine in platelet cell membrane flip-flops from the inner to outside surface of the bilayer. a. This new surface is essential for proper coagulation; clotting factors in plasma now can localize together on activated platelet.

RTK (Receptor Tyrosine Kinase) Activation

1. EGF binds to EGFR 2. EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity. 3. Autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of EGFR occurs. 4. Further cascades take place

OVERVIEW of ENZYME MECHANISM

1. Enzymes make the transition state 2. Vast majority of interactions between the substrate and amino acids' active site are NONCOVALENT 3. Conformational shape change during catalysis 4. Some use cofactors

Regulation of Coagulation (steps 1 & 2)

1. Escaped Thrombin is inactivated by antithrombin III inhibitor that is bound to heparin on endothelial cells of intact blood vessels. 2. Thrombin that escapes the clot binds to thrombomodulin of intact endothelial cell surface. a. This regulatory protein changes thrombin's proteolytic specificity so now thrombin activates protein C in the plasma. b. Activated protein C & protein S together degrade Factors Va, VIIIa of the clotting cascade.

RELEVANCE OF FIBROUS PROTEINS

1. Essential and very abundant proteins in the body. 2. Proteins with regular secondary structural elements that form elongated fibers that add strength or elasticity to tissue. 3. Collagen and elastin are very abundant in: a) skin and bones b) connective tissue, tendons, ligaments c) blood vessels 4. Keratin is main component of hair, nails, and outer epidermis. 5. When these fibers are defective or damaged, tissues have different physical properties that lead to maladies such as: scurvy, Ehlers- Danlos Syndrome, emphysema.

CONTROL OF ENZYME ACTIVITY

1. Fast (virtually immediate to seconds time scale) 2. Intermediate (minute time scale) a. Covalent modifications (e.g. phosphorylation by kinase). b. Binding to modulator proteins (e.g. cAMP regulatory subunit). c. Zymogen activation (e.g. clip by a protease to make active form). 3. Slow (hour to day time scales)

How do you transport a gas?

1. Globin Proteins w/ heme complex 2. Hb binds oxygen in the lung (the high O2 concentration or high partial pressures = pO2) and releases in tissues (the lower O2 concentration). 3. Mb binds O2 when tissue is well oxygenated and released as the pO2 in tissues falls substantially (during exertion, etc.).

Steps in Tissue Pathology

1. Gross in and preserve specimen (gross pathology) 2. Prepare block and slide 3. Stain and visualize (histopathology) 4. Prepare special studies

PIP2 Pathway

1. Hormone-receptor activates Phospholipase C 2. PLC hydrolyzes PIP2 Phosphatidylinositol-4,5-bisphosphate into DAG: Diacylglycerol & IP3: Inositoltrisphosphate 3. DAG remains on surface and activates Protein Kinase C leading to many varied cellular responses 4. IP3 activates IP3-sensitive Ca2+ channels in ER allowing Ca2+ ions into cytosol

Genetic Defects of HB (Abnormal O2 Transport)

1. Increased Affinity 2. Decreased affinity leads to normochromic anemia. 3. Oxidation of heme (HbM).

Stepwise process of malignancy

1. Initiation 2. Latency 3. Precancer 4. Cancer 5. Progression (aneuploidy, tumor heterogeneity, aggressiveness, metastasis, dormancy, accelerated tumor growth)

DIAGNOSTIC USE OF ENZYMES

1. Measure levels of enzymes in bodily fluids and compare to the "healthy" levels or ranges. Common tests utilize: a. Enzyme assay - utilize specific substrates and buffer to select and to detect enzymes in a complex mixture. b. Electrophoresis - separate various enzymes by charge and identify the different isozymes. c. Immunochemistry - use selective antibodies to detect and quantify an enzyme (often use an enzyme-based reporter group). 2. Benefit: quick and specific assays with minimal invasiveness.

Why are enzymes important in medicine?

1. Most drugs are enzyme inhibitors! 2. Bad or missing enzymes can cause problems! a. Tay-Sachs b. PKU c. Acute Pancreatitis

ALLOSTERIC ENZYMES AND REGULATION (how are enzymes in the body fine-tuned)

1. Multimeric enzymes can "communicate" (in analogy to Hemoglobin) between separate active sites to regulate activity. 2. Allosteric enzymes can "sense" the level of its product, a downstream metabolite or an upstream precursor. 3. Integrated metabolism utilizes these features of cooperativity extensively. * Allosteric enzymes do not obey typical kinetics; their Michaelis-Menten plots are sigmoidal curves (not hyperbolic).

ENZYME OVERVIEW

1. Names usually end with -ase. 2. Enzymes can employ many types of simple reactions (acid/base, H abstraction/donation) to synthesize or to break down molecules. 3. Lower the Free Energy of Activation (∆G++) so they decrease time to reach equilibrium (10^6 to 10^12-fold faster). 4. Do not change the equilibrium constant (Keq) or the Standard Free energy ( ∆G) of the reaction. 5. Can couple favorable reactions to unfavorable reactions to drive reaction (e.g. utilize the energy of ATP to create another molecule).

FIBRIN CLOT REMOVAL

1. Plasmin protease cleaves fibrin at flexible triple chain rods between the D and the E domains. 2. Formation of active plasmin - a) Tissue plasminogen activator (tPA) and plasminogen zymogen bind to the clot network and form a complex. b) Plasminogen is then proteolytically clipped by tPA to form active plasmin. c) The plasmin cleaves fibrin. 3. Antiplasmin in the plasma inhibits any plasmin released from clot.

KERATIN

1. Primary structure - Tandem repeats of 7 AAs (the equivalent of two turns of an alpha-helix) that form a nonpolar and a polar surface. 2. From micro to macroscale => alpha helix, protofibril, microfibril, macrofibril of hair. 3. Many cysteines that are cross-linked so that keratin fibrils are insoluble and resistant to stretching (permanent waves undo Cys cross-links).

OVERVIEW of COLLAGEN

1. Primary structure - mostly GPX sequence repeats, ~1000 AAs. 2. Forms triple helix of three intertwined, open left-hand polypeptide helices. 3. Special Features: - Small glycine residue > helices wrap together closely. - Peptide bonds are perpendicular to the chain axis; allows the carbonyl to point to other adjacent chains. - Triple helices self-assemble into fibrils that reinforce tissues.

2 major modes of Proteoltyic cleavage

1. Removal of a short peptide from the N- or C- terminal region of a polypeptide, leaving a shortened molecule that folds into the active protein. (simple chopping) 2. Cleavage of polypeptides into segments generating more than one active protein. (complicated patterns)

FIBRIN CLOT FORMATION (1-2)

1. Structure of Fibrinogen - three domains, DED, connected by flexible triple chain rods - 6 polypeptides - very soluble. 2. Aggregation - Platelets bind fibrinogen D domains and form crosslinks between platelets .

The TGF-beta Signaling Pathway

1. TGF-β dimers bind to a type II receptor which recruits and phosphorylates a type I receptor. 2. Type I receptor then recruits and phosphorylates a receptor regulated SMAD2. 3. The SMAD2 then binds to the common SMAD (coSMAD) SMAD4 and forms a heterodimeric complex. 4. This complex then enters the cell nucleus where it acts as a transcription factor for various genes, which trigger angiogensis, cell growth, cell mobility, arrested growth and apoptosis.

Thrombin Production (Step 1)

1. Upon wounding and platelet activation, prothrombin is localized to the activated platelet surface . A unique modification, the addition of gamma-carboxyglutamate residues (Vitamin K-cofactor dependent carboxylation of glutamate in the liver), allows the zymogen to bind.

MEDICAL RELEVANCE OF ENZYMES

1. Without catalysts, biological reactions would be too slow at body temperature and/or not selective enough. 2. Defective enzymes cause deficiencies including storage diseases (Tay-Sachs) or metabolic blocks (phenylketonuria) 3. Enzymes that are not controlled or regulated properly cause problems such as destruction of cell & tissue structures (pancreatitis) or loss of required fine-tuning of reactions (hypercoagulation). 4. Diagnostics - noninvasive method for determining pathology (Did Grandpa really have a heart attack?).

Medical Relevence

1. Your body requires about 500g O2/day, but only 30g/day can be physically carried by water/plasma alone. a. Hemoglobin (Hb) allows higher delivery in blood. b. Myoglobin (Mb) is a reservoir for O2 in muscle cells (diving whales). 2. Defective Hb causes many problems including sickle cell, cyanosis, hemolytic anemia. 3. Understanding Hb structure/function allows the treatment of defects and certain types of poisoning.

Rods & Rhodopsin

1. light causes conformational change in rhodopsin 2. Rhodopsin activates G-protein Transducin 3. Active Transducin (w/ GTP) activates cGMP Phosphodiesterase 4. Phosphodiesterase hydrolyzes cGMP 5. cGMP can no longer activate cation channels

Nuclear localization of proteins pathway

1. protein w/ NLS binds to importin-alpha 2. protein-importain complex binds to importin beta 3. complex enters nucleus through pore 4. comples dissasociates inside after binding with GTP

Upon oxygen binding to a heme:

1. the bond between the Fe atom and the coordinating Ns shortens . 2. Fe is pulled into plane of heme group. 3. F helix is pulled, and protein shape changes to R (relaxed) form. 4. The subunit interface pocket becomes more compact and more hydrophobic. 5. pka of some residues participating in ionic bond network changes: a. Positive His group was stabilized by a nearby negative Asp group [forms ionic bond, too] in aqueous phase, but not organic phase. b. His residue sheds a proton to get neutral. 6. Ionic bonds in pocket between subunits break, causing other subunits to shift toward R form which binds O2 150-300X more tightly than T form (positive cooperativity). 7. The alpha-beta pairs shift with respect to each other.

Question: What is the chance for recurrence for disorders caused by mitochondrial mutations?

100%

What would the consequence be for the offspring of a couple, if one of the partners has a balanced 21;21 Robertsonian translocation?

100%!!!

Prader Willi Syndrome (PWS)

15q11.2-q13 Maternal snoRNA genes imprinted (repressed)Paternal snoRNA normally expressed - if deleted => PWS

Factor XA production cascade

2 ways to make Factor Xa from X zymogen 1. extrinsic pathway - needs tissue factor (MAIN CLOT INITIATOR) 2. intrinsic pathway - needs RNA from damaged cells (SUPPORTIVE)

FIBRIN CLOT FORMATION (3-5)

3. Proteolysis and Activation - Thrombin cleaves off A & B fibrinopeptides > exposes binding domain E which interacts with D domains of other fibrin molecules. (fibrinopeptides have a high negative charge and keep fibrinogen from interacting) 4. Polymerization - Formation of a staggered array of monomers to form fibrous aggregates. 5. Crosslinking - Thrombin activates transglutaminase, an enzyme that forms bridges between D/D & D/E domains > stabilizes the clot.

Regulation of Coagulation (step 3)

3. Thromboxane (TxA2) from activated platelets stimulates healthy intact endothelial cells to produce the small molecule prostaglandin I2 (PGI2) that inhibits platelet activation.

polyploidy

3n, 4n, 5n,.....

Turner syndrome

45, X_

Klinefelter

47, XXY

Fragile X (Premutation alleles)

55-200 repeats Unstable and prone to expansion Possible toxic effect of mRNA with expansion Females with premutation 15-20% premature ovarian failure May develop FXTAS (Fragile X-associated tremor/ataxia syndrome) Males with premutation Normal range of intelligence 1 in 3 FXTAX after age 50 years

Stroke

= brain attack

splice site mutation

=skipping of exons via mis-splicing "Mutations in intron splice sites flanking an exon usually result in skipping of that exon in mRNA" -often results in premature termination of translation

Answer for Question

A - I

Mendelian Diseases

A Mendelian disease results from a single mutant gene and is often inherited in a simple recognizable pattern Autosomal or X-linked "dominant" if phenotypically expressed in heterozygotes and "recessive" if clinically manifest in homozygotes The type of Mendelian disease is deduced by constructing a pedigree and analyzing the pattern of transmission of the trait within the family

Basement membrane

A composite of several large glycoproteins that form an organized scaffold to provide structural support to the tissue and also offer functional input to modulate cellular function

ACS (definition)

A new infiltrate on CXR accompanied by at least one of the following:- chest pain fever hypoxia respiratory diseases (SOB - shortness of breath , wheezing, cough)

replicon

A nucleic acid molecule, or part of one, that replicates as a unit, beginning at a specific site within it.

Collagen Role - WOUND HEALING

A). Scar tissue consists mostly collagen [ex. liver cirrhosis; after damage from virus, alcohol or drugs, the dead cells are replaced with collagen fibers]. B). Tissue remodeling and repair requires controlled collagen degradation. C). In infection, the abscess is normally walled off with collagen to contain the microbes, but some bacteria secrete collagenases to digest the protective barrier & escape into the surrounding tissue!!

Protein Entry into Cells

A. Phagocytosis: B. Pinocytosis: C. Receptor-Mediated Endocytosis:

Treatment of Stroke

ACUTE: - Exchange transfusion to decrease Hgb S to <30% - Rehabilitation if necessary CHRONIC: - Chronic transfusion to prevent production of sickle cells - indefinite duration - Chelation therapy to prevent Fe overload PREVENTION: - Transcranial Doppler screening

Protruding N-terminal tails of histones

Acetylation = Active Methylation = Inactive

Other Issues to be Monitored

Acquired hypothyroidism Atlanto-axial subluxation (Paralympics!) Obesity Celiac disease Obstructive sleep apnea Seizures

Chemical Modification of Proteins

Addition of small chemical groups Addition of sugar side chains Addition of fatty acid side chains Addition of peptide groups Modification of amino acids within the protein

Agonists and Antagonists

Agonists- Dexamethasone (GR): anti-inflammation Antagonists- Mifepristone/RU-486 (PR): medical abortion Selective Receptor Modulators (tissue-specific): SERM: Tamoxifen -antagonist in breast -agonist in uterus and bone

Papanicolaou (Pap) Smear

Detection of atypical cervical epithelial cells associated with cervical cancer risk.

pH of a solution affects the charge of weak acids & bases differentially

low pH - high protons high pH - low protons

Single Gene Disorders

Autosomal dominant Autosomal recessive X-linked Y-linked

Zellweger syndrome

Autosomal recessive disease associated with the absence of peroxisomes. Fatal by the age of 6 months

Spine Fusion Surgery

BMP-2 in sponge of collagen No hip surgery for bone grafts required Recent controversies (unwanted bone growth, cancer)

Brachydactyly

BMPR1B mutations

Signaling Is Everywhere

Bacteria: e.g., gastrointestinal tract (signal each other and with epithelial and immune cells) Multicellular organisms: >2 billion years ago in cyanobacteria >500 million years ago: plants/ animals

Basement membrane:

Basal lamina: made by epithelial cells (Type IV collagen, laminin). Reticular lamina: made by CT fibroblasts. (Type III collagen. & Blends with underlying fibers.)

Chromosome Micro Analysis

CMA -array based genomic hybridization methodology that allows for simultaneous analysis of the entire genome for a large number of genetic disorders -with a single test, CMA can identify UNBALANCED abnormalities that are not detectable by routine chromosome analysis or FISH analysis -limited by the number and spacing of probes on the micraarray

Choristoma

non-neoplastic Tissue that is out of place (e.g. pancreas in stomach)

Epithelium (tissue origin)

Carcinoma

Cilia

Composed of stable microtubules (tubulin) -9+2 arrangement -Longer than microvilli

DNA to Chromosome

DNA => Double Helix => Nucleosomes w/ spacers => chromatin fiber => scaffold => loops of chromatin => sister chromatids w/ centromere

C Banding reveals constitutive heterochromatin

Constitutive - always inactive (e.g. all centromeres and 1q, 9q, 16q)

mutation hotspot

CpG - most common site for mutation causes

Collagen Pathway

DNA=>mRNA=>preprocollagen=>procollagen=>tropocollagen=>collagen (lysl oxidase)

Ubiquitin-Proteasome System

Degradation of Intracellular Proteins: - Damaged proteins - Unfolded - Chemically altered - Naturally short-lived species

Clinical pathology

Diagnosis of disease by analysis of blood and other human biospecimens (e.g. blood glucose)

Autopsy Pathology (Forensic)

Diagnosis of disease in deceased patients by gross examination, histomorphology, immunohistochemistry, and other methods

Nuclear-encoded mitochondrial diseases:

Diseases of mtDNA stability: - mtDNA multigene deletions - depletion (decreased mtDNA content) - (non-structural, non-Complex diseases)

Achondroplasia

Disproportionate short stature Macrocephaly Frontal bossing Hydrocephalus Mild hypotonia Cervical cord compression Trident hand Autosomal dominant Chromosome 4p FGFR3 gene

Protein Translocator

Donut-shaped transmembrane protein of the RER, forms a pore in the ER membrane; pore allows the growing polypeptide chain to enter the ER.

Nucleus-encoded mitochondrial genes:

Dozens for electron transport chain subunits Others encode assembly proteins for complexes, transcription, translation, multiple other processes

GI Complications

Duodenal atresia or stenosis Hirschsprung disease (<1%) Tracheo-esophageal fistula Imperforate anus

Life Expectancy in Sickle Cell Disease

Early 1970s - Median age of survival - 16 years overall Mid 1990s - Median age of survival Sickle Cell Disease-SS 40-45 years Sickle Cell-Disease-SC 60-65 years

alpha-1 antitrypsin deficiency & Lungs

Elastin Turn-over & Lung Disease>SMOKING

Phenylketonuria (PKU)

Error in gene for phenylalanine hydroxylase (PAH) Untreated deterioration of intellect onset of seizures Newborn screen post 24 hours Diet for life Strict dietary management for pregnant women with PKU

Loss of APC can lead to familial colon cancer

Familial adenomatous polyposis (FAP) • colon carpeted with polyps (c.f.,HNPCC) • autosomal dominant • APC (adenomatous polyposis coli;5q21) :tumor suppressor - causes missense mutations and deletions) • colorectal cancer risk = 100% at 50 years • tx:prophylactic colectomy

Granulocyte maturation (overall trend)

From larger to smaller size. Round, fine nucleus, to dark, segmented nucleus. Increasing cytoplasm. No granules to primary granules (lysosomes) to specific granules.

Secondary structure (alpha helix)

r-group stick out to the side; all possible good hydrogen bonds are formed...

Cytopathology

Findings from the microscopy examination of individual cells of groups of cells

Gross Pathology

Findings from the unmagnified (naked eye) examination of specimens

Neurofibromatosis 1

First degree affected family member Six or more café-au-lait spots Two or more neurofibromas or one plexiform neurofibroma Lisch nodules Optic glioma Pseudoarthrosis

Relatives Risk - Genetics of multifactorial / complex diseases

First, second and third degree relatives share 1/2, 1/4 and 1/8 of their genes, respectively. The frequency of multifactorial diseases reflects this sharing of genes and is an important determinant of recurrence risk within families.

Tissue Preparation: lipids extracted during dehydration by solvents

Fixed, dehydrated tissue embedded in warm paraffin => Tissue blocks sectioned on microtome => parafin sections placed on glass slides => de-waxed sections stained and cover-slipped - FDESS - FIXATION, DEHYDRATION, EMBEDDING, SECTIONING & STAINING

Robertsonian Translocation (Gamete)

Gamete formation, and consequence for offspring

Factors affecting the Hardy-Weinberg equilibrium

HWE is assumed to exist in most large populations

Terms to know

Genetic disease, mitochondrial & nuclear DNA, chromosome, gene, Purines & pyrimidines, nucleotide, double helix, 5' and 3', Nucleosome, chromatin, centromere, kinetochore, telomere, aneuploidy, translocation, Polymorphism, repetitive DNA, copy number variation (CNV), single nucleotide polymorphism (SNP), Euchromatin and heterochromatin, histone deacetylation, CpG dinucleotide, DNA methylation

G-banding

Giemsa stain - the most common stain technique used to identify individual chromosomes

Glycoproteins

Glycoproteins typically consist of 10-50% carbohydrate compared to proteoglycans consisting of over 95% carbohydrate.

HAMARTOMAS

H hamartomas on skin and in CNS A adenoma sebaceum M mitral regurgitation A ash-leaf spots R rhabdomyoma T tuberous sclerosis O autOsomal dominant M mental retardation A angiomyolipoma S seizures, subependymal giant cell astrocytomas

How do you optimize oxygenation of tissues? (fine-tuning of oxygen deliver)

Hb affected by: a. Oxygen concentration / positive effect at high pO2 b. pH / negative effect at low pH c. Carbon dioxide concentration / negative effect at high conc. d. 2,3-bisphosphoglycerate (BPG or DPG) / negative effect - These factors alter quaternary structure of Hb (not the Mb monomer) and optimize 02 delivery in the body (deliver 65% rather than the predicted 24-36% of oxygen load if Hb were like Mb).

Germline mutations in mismatch repair genes cause HNPCC

Hereditary non-polyposis colon cancer (HNPCC; Lynch syndrome) • 1/1,000 people • all colorectal cancers:3% • autosomal dominant • MLH1,MSH2,MSH6,PMS2 • Colorectal cancer risk:80%;Xage = 44 • Other cancers:endometrial, stomach, ovarian...

Females may manifest an X-linked disease

Heterozygosity Cells display 'all or none' phenomenon, due to random X-inactivation Skewed X-inactivation Turner syndrome (45,X) X-autosome translocation with inactivation of the normal chromosome Homozygosity / Compound heterozygosity Due to high prevalence of mutations in a population, a female may be homozygous for an X-linked recessive mutation, e.g. G6PD deficiency in African-Americans (10% of males have G6PD deficiency)

repeat expansion

Huntington example-excessive repeats of a triplet codon within the gene. Normal people have these repeats also but the count of repeats remains below threshold.

Disorders with mitochondrial inheritance

Kearns-Sayre syndrome MERFF MELAS mitochondrial DNA depletion syndrome NARP Pearson syndrome

Fluorescense in situ hybridization

INSERT SLIDE 43

CMA

INSERT SLIDE 48

Parametric (standard) linkage analysis

Identify rare families with near mendelian inheritance (BRCA1 and BRCA2)

Recombination and the concept of linkage: Lack of independent assortment between linked loci

Image on right - close they are on the same chromosome, the less likely they are to be separated after recombination due to crossing-over errors Image on left- Loci on same chromosome, but far apart enough that they are treated as if they are on separate chromosomes

How does a protein fold?

In a polypeptide chain, the only rotation is around the alpha -carbon. Due to steric hindrance and/or interactions between R-groups, only certain favorable angles are allowed between any two residues.

Uniparental Disomy Disorders

PWS AS Beckwith-Wiedemann syndrome (BWS) Silver-Russell syndrome UPD 14 Rarely AR disorders

Reverse Signalling

JAG (Jagged) 1, 2 DLL (delta-like) -1, -3, -4 Physiological importance less studied/clear

Mitogen-Activated Protein Kinases (MAPKs)

Just know Stimulus => MAPKKK => MAPKK => MAPK => Biological Response

X-Linked

Incidence in males much higher than females Heterozygous females may express the condition depending on pattern of X inactivation Carrier females: 50% of sons affected, 50% of daughters are carriers Affected males: all sons unaffected, all daughters are carriers (no male to male transmission) Affected males are typically related through females Isolated cases usually due to new mutation Recurrence to non-carrier females may be due to germline mosaicism

Fragile X Syndrome (cytogenetic)

Initial recognition of the syndrome Fragile site at Xq27 In cell culture, folate - or thymidine + produces fragile site

Macular adherens

Intercellular adhesion via "spot" attachments, Cadherin proteins attach to each other extracellularly, and plaques intracellularly (restricted to epithelial and few other cells). Keratin anchored to cytoplasmic plaques

Erythrocyte maturation (overall trend)

Large, pale nucleus to darker, smaller nucleus. Loss of nucleus. Increase in cytoplasm. Decrease in size. Cytoplasm changes from blue (RNA) to grayish (RNA and hemoglobin) to red (hemoglobin; no RNA)

G Protein-Coupled Receptors (GPCRs)

Largest family of cell-surface receptors Serpentine/seven-transmembrane domain receptors ~30% of all drugs target GPCRs Ex - Rhodopsin, taste & odorant receptors, chemokine receptors, serotonin, dopamine, glutamate, histamine, sympathetic & parasympathetic

Blood (tissue origin)

Leukemia

BAC (bacterial artificial chromosome) vectors

Libraries of the human genome, fragmented and cloned, are inserted in these for storage

NOTCH Receptors

Ligand binding induces cleavage by TACE (TNFalpha-converting enzyme) and then gamma-secretase

Secondary structure (beta sheet)

r-groups stick out of or into the page.. same thing w/ hydrogen bonds

Non-Parametric (model-free) linkage analysis

Look for alleles / chromosomal regions shared among affected relatives (Identity by descent) e.g. Sib-pair analysis (e.g. Type 1 Diabetes)

Lymphoid tissue (tissue origin)

Lymphoma

Desmosome

Macula Adherens, Cadherin attached to plaques, intracellular keratin

Clinical significance of mitochondrial gene mutations

Maternal Inheritance Heteroplasmy

Two Distinct Proteins for Oxygen Delivery

Mb - hyperbolic Hb- sigmoidal

Immunoreactive trypsinogen (IRT)

Measurement in blood of newborn babies is an assay in rapidly increasing use as a screening test for cystic fibrosis

Zonula Adherens

Mechanical attachment between neighboring cells, Cadherin proteins attach to eachother extracellularly and attach to cytoplasmic proteins anchored to filamentous actin (Cadherin attached to Actin "belt")

X-Linked Recessive Inheritance

Mechanism similar to autosomal recessive-due to loss of function Women can be affect-X linked activation-unusual cases

RT-PCR & Northern Blot

Medical Application: Analysis of gene expression levels PROCESS: -RNA fragments -run on gel -blot to membrane -hybridize with specific probe -uses X-ray film

Autosomal dominant inheritance (breast/ovarian cancer - BRCA1 or BRCA2) with gender-bias expression

Men can pass down breast cancer traits too—for example in this pedigree, the affected male is a carrier who passes it down to his daughter

Centrisome

Microtubule organizing center -2 centrioles -formed from nine sets of microtubules -triplet @ proximal into doublet @ distal -attached to eachother by flexible linker

Mitochondrial Inheritance

Mitochondrial inheritance is NON-MENDELIAN pathways. Mitochondrial DNA transmitted from mother-so all mother's offpsring are affected. Male offspring never transmit disease Variability exists in heteroplasmy There are multiple copies of mitochondria in cell in they mostly all the same Homoplasmy—all the mitochondria can be normal or abnormal Heteroplasmy—partial affected.

Fragile X Syndrome

Most common type mental retardation in males 1/1250 males Mild to profound mental retardation Macrocephaly Prognathism Large ears Macroorchidism Hand flapping Poor eye contact X-linked

Marfan syndrome

Myopia, retinal detachment Dural ectasia Dolicocephaly, high palate, crowded teeth Decreased subcutaneous fat Mitral valve prolapse Joint laxity, scoliosis Tall stature Aortic root dilation Arachnodactyly Ectopia lentis Autosomal dominant Chromosome 15q Fibrillin gene

Euchromatin

Most of the DNA in interphase nuclei; in extended, active form

X-Linked

Mother could be a carrier of the same condition, Factor VIII deficiency Daughter could have Turner syndrome Daughter could have a X;autosome translocation

addition of sugar side chains

N-linked glycosylation, O-linked glycosylation

Cytoskeleton

Network of protein filaments that extend throughout the cytoplasm of eukaryotic cells -organize intracellular components -adopt a variety of shapes -carry out coordinated movements -provide mechanical support

Neoplasm

New growth, can be benign or malignant

Cystic Fibrosis (IRT)

Newborn screening protocol on filter paper blood spot IRT >65 reflex to DNA panel one or two mutations identified reflex to sweat test < 30 mmol/L => unaffected with CF > 60 mmol/L => affected with CF 30-60 mmol/L borderline, retest in four weeks abnormal sweat test refer to CF clinic

Challenges with Genetic mapping of multifactorial / complex diseases

No specific pattern of inheritance Reduced penetrance Diagnostic criteria not well established

Colorectal carcinoma can arise in a stepwise fashion

Normal => adenomatous (w/hyper-chromatic stratified nuclei) => tubular adenoma (polyp) Bert Vogelstein produced model of multistep carcinogenesis

Recurrence risk of multifactorial traits depends on....

Not all affected first degree relatives are equal

Chance of Recurrence (multifactorial)

One child affected => 4% One parent affected => 4% Parent and one child affected => 17% Two children affected => 9%

The diseases

Osteogenesis imperfecta: glycine point mutation prevents formation of triple helix (think zipper analogy) Scurvy: vitamin c deficiency, the prolyl hydroxylase cannot function so you're triple helix is not stabilized. If its not stabilized then, it melts at body temp. Ehler Danos: Happens if your copper deficient, if your lysyl hydroxylase doesnt function properly. Leads to failure of cross-linking of monomers.

Hamartoma

Overgrowth of normal tissue

Complex Response

P-Tyr binding proteins -Adaptors (Grb2, Shc) -Enzymes (PI3K, Src, PLCgamma)

detects small (<1kb) mutations

PCR

mutation detection

PCR, dot blot, restriction digestion, southern, MLPA, microarrays, DNA Sequencing (Sanger & "nextgen") *All single-base substitutions are detected by a combination of PCR and the following techniques: -known: restriction digestion, dot blot, (sequencing) -unknown: DNA sequencing All mutations are ultimately confirmed by DNA sequencing

Robertsonian Translocation

Parent with Robertsonian translocation (involving D (13,14,15) or G (21, 22) chromosomes), but not t(21;21)

Autosomal Recessive

Parents are asymptomatic carriers of the condition 25% chance of recurrence in siblings Equal number of males and females affected Horizontal transmission Consanguinity is possible, especially in very rare conditions Cystic Fibrosis,

Angelman Syndrome

Paternal UBE3A is imprinted (repressed) in the brain Maternal UBE3A is normally expressed - if deleted AS - 70% of cases: segment of the maternal chromosome 15 containing this gene is deleted. - 11%: Angelman syndrome results from mutations within the UBE3A gene itself. Most of these mutations lead to the production of an abnormally short, nonfunctional version of ubiquitin protein ligase E3A. - Because the copy of the gene inherited from a person's father (the paternal copy) is normally inactive in some areas of the brain, loss of the maternal copy prevents any of the enzyme from being produced in these brain regions. This lack of enzyme function likely causes the major signs and symptoms of Angelman syndrome.

Clues to identify modes of inheritance

Patients in single OR multiple generations males < > females OR males = females mother < > father OR maternal = paternal

Preprotein

Protein with a secretory signal attached

PCR

Polymerase Chain Reaction

Common Types of DNA Polymorphism

Polymorphisms that are relatively harmless Polymorphic markers that are found outside of genes Inherited from father More likely to be informative Fig C - SNP - change between an A and a C..

Gene testing

Presymptomatic testing - When there is a family history of HD - Informed consent (usually 2 counseling sessions) - Not common; only 20% request such testing Confirmation of clinical diagnosis Prenatal testing - It may be combined presymptomatic / prenatal testing (i.e. the mother may not know her own status) - Ethical issues - parent's versus child's right to know & potential for insurance / career discrimination) - Preimplantation diagnosis

Kartagener's syndrome

Rare congenital disorder in which cilia are non-functioning (dyein arms or other component missing). Results in infertility and chronic bronchitis

Intermediate filaments

Ropelike fibers that provide structural support and cell attachment sites -Made of intermediate filament proteins (e.g. neurofilaments, feratin, vimentin) ex: Keratin filaments anchor desmosomes

Connective Tissue (tissue origin)

Sarcoma

Cystic Fibrosis (cytology)

Thousands of deleterious mutations Different labs have different standard mutations, most ~92-95% of total Some mutations familial Some mutations are mild If negative on standard panel, chance of being a carrier 1/250 ish

Autosomal dominant inheritance (Neurofibromatosis type I) with variable expression

Severity may vary on the affected = Variable Expression = expressivity ---- the degree of expression a and b fully affected; c partially affected

Lifetime Take Home

Signaling is crucial in development, homeostasis and disease formation Targeting signaling pathways is an effective way of clinical care

Signalling pathway

Signaling modulates everything, including growth, differentiation, mobility, immune responses

detects large (>1kb) mutations

Southern blot INSERT SLIDE 83

Down Syndrome Critical Region

Specific genes on chr21 are thought to be responsible, through modulation of developmental pathways, for specific aspects of the abnormal genotype Located in a region on 21q, known as Down syndrome critical region (or DSCR, 21q22 to qter)

Smooth muscle

Spindle-shaped (tapered ends) with centrally located nucleus. Thin actin filaments insert onto dense bodies. Contraction stimulated by endocytosis of calcium vesicles and SER. Communicate with neighboring fibers via gap junctions. High regenerative capacity in response to injury

Receptor Tyrosine Kinases

Stop Sign

cardiac muscle

Striated branching muscle cells. Myocardium of heart. Rhythmic contraction coordinated via gap junctions.

Williams Syndrome

Supravalvular aortic stenosis, IQ ranges 41 - 80, Hoarse voice, Friendly, outgoing personality, Stellate irides, FTT in infancy, Microdeletion involving elastin gene (chromosome 7q11.2), Autosomal dominant, Mild microcephaly, Depressed nasal bridge, Large mouth, full lips, Hypoplastic nails, Prominent earlobes, Renal anomalies, Joint laxity, hypercalcemia

Smooth Endoplasmic Reticulum

Synthesized steroids, contain enzymes that detox -Important in the muscles (store calcium)

Hb Subunit Interactions

T form (taut) - low pH, high CO2 and high 2,3 BPG favor this form & has lower affinity for O2 R Form (relaxed) - high pH, low CO2, or low 2,3 BPG favor this form & has higher affinity for O2

Cancer Mutations in TGF- Pathway

TGF-beta type II receptor: Colon and gastric cancer Smad4: Pancreatic (50%) and colorectal tumors (15%) Smad2: Colon cancer

cAMP-Dependent Processes

TSH, LH, Adrenaline, Glucagon,Vassopressin

Appearance of 3-Dimensional Structures in 2 Dimensions

The appearance of histological structures are affected by their orientation within the plane of section.

Metastatic Disease

The cause of most cancer deaths, not the primary tumor (blood, lymphatic system, or peritoneal cavity, also nerves)

TERMS TO KNOW

The central dogma, transcription, RNA processing, splicing, Translation, genetic code and codon, initiation and STOP codons, degeneracy of the genetic code, Gene structure, alternative splicing, tissue‐specific expression, Imprinting, euchromatin, heterochromatin, X-inactivation, Mitochondrial genome, variant genetic code, maternal inheritance, homoplasmy, heteroplasmy

Imatinib

targeted molecular therapy, a tyrosine kinase inhibitor (indicated by -ib)

Genotype

The genetic component of an individual Commonly used to refer to alleles at a specific locus (gene)

Most common site for metastatic disease

The liver

Phenotype

The observable characteristics of a cell or organism, e.g. trait or disease

genetic anticipation

The occurrence of a genetic disorder at progressively earlier age in successive generations. -disease severity is proportional to the length of the repeat expansion -expanded repeats are unstable and they may increase in length from one generation to the next ex: myotonic dystrophy pedigree

Carcinogenesis

The process by which cells acquire attributes that confer a malignant phenotype. note:applies to all cancers, not just carcinomas - accumulation of mutations by normal cells over the course of time

Exocytosis

The vesicular transport of material from the trans-Golgi network to the cell surface -Constitutive secretory pathway (default -Regulated secretory pathway

The CAG repeat is unstable in the male germline

This explains why: - Anticipation is mainly seen on paternal transmission - Juvenile HD is mainly seen following paternal transmission - Intermediate alleles (29-39) expand during paternal transmission

Junctional complex

This is a combination of junction that often join together epithelial cells

Autosomal dominant inheritance

Why would a mutation in one allele cause problems for the organism? Various possibilities exist: Haploinsufficiency Dominant-negative effect Gain-of-function

Fragile Sites Cause chromosome breakage and rearrangements

Throughout genome Chromosomal locations Related disorders/disease: Fragile X syndrome Fragile XE leukemias tumorigenesis

Zonula Occludens

Tight Junction, provides paracellular barrier, completely encircles cell

Filament Type: Keratin

Tissue Origin: Epithelial Cells

Filament Type: GFAP

Tissue Origin: Glial Cells

Filament type: Vimentin

Tissue Origin: Mesenchymal cells

Filament Type: Desmin

Tissue Origin: Muscle Cells

Filament Type: Neurofilament

Tissue Origin: Neurons or Neural crest

Directionality of DNA

Transcription occurs in the 5' to 3' direction

clot stabilization

Transglutaminase crosslinks the glutamine and lysines of fibrin (within 8-10 min).

1. Enzymes make the transition state

Transition state - the fleeting intermediate between substrate and product, appear more often in a reaction mixture with the active site (residues involved in catalysis). a. Enzymes stabilize the transition state by forming favorable interactions with active site. b. An alternative reaction pathway with a lower energy barrier is utilized.

Patau syndrome

Trisomy 13 cleft palate common extra toes or fingers frequently die within first days of life 5-10% of children with this condition live past the first year

Protein Degradation

Two major organelles are involved in the degradation of proteins within the cell A. Lysosome: - Extracellular proteins - Intracellular proteins within organelles B. Proteosome: - Intracellular proteins

Pedigree suggesting mtDNA defect:

Unusual maternal inheritance pattern (purple): - Multigenerational - Passed only by women - Can be passed to ALL offspring - PLUS additional features (slides to follow):

Factors affecting the manifestation of mitochondrial mutations

Variable number of mitochondria per cell (2-100) and mtDNA chromosomes per mitochondrion (5-10) Heteroplasmy: Different cells / tissues may have varying numbers of mutant mtDNA Variable susceptibility of different tissues to defective oxidative phosphorylation caused by abnormal mitochondrial function

VEGF

Vascular endothelial growth factor -most important, key activator for tumor to start inhibitor: Bevacizumab (monoclonal antibody)

structural cytogenetic mutation

deletion duplication insertion inversion translocation -reciprocal -robertsonian microdeletion

skeletal muscle

Voluntary movements. Described as fibers. Multinucleate syncytium. 10-100 micron diameter. 1 mm-30 cm length. Vertical striations represent the highly level of contractile myofilament organization.

WNT Pathway

WNT: Morphogen (secreted by glycoprotein - 19 members in humans) APC _ Adenomatous Polyposis Colli

restriction digestion

We skipped it..

7.5

What % of all conceptions have chromosome abnormalities

0.5

What % of live births have chromosome abnormalities

Cloning

What is DNA cloning? A: -A way of 'capturing and copying' a specific DNA fragment (gene of interest) -Large quantity of this DNA fragment is then available for further analysis (e.g., DNA sequencing) and manipulation (e.g., recombinant gene expression) Recombinant DNA tech and PCR allow for isolation & amplification of DNA sequence from gene of interest

balanced translocation

When two chromosome ends break off and both reattach into the genome (don't remain floating around in the nucleus where they would not be duplicated or divided between both cells) -no net gain or loss of genetic material

WAGR

deletion of 11p13 deletion including WT1 and PAX6 genes -wilms tumor, aniridia, genital anomalies, growth retardation

Tight Junction

Zonula occludens, provides paracellular barrier

Cancer

a common consequence of defective DNA repair

Adenocarcinoma

a majority of cancers of the colon (cancer of the epithelium originating in glandular tissue)

vector

a replicon that makes multiple copies of itself(clones) when introduced into a host bacterial cell (via transformation)

Angelman

deletion of maternal 15q11-q13

recombinant vector

a vector that has a DNA fragment inserted into it.

Autosomal dominant inheritance (Marfan syndrome) with reduced penetrance

a) Clinically unaffected b-e) Marfan syndrome Not everyone with a fibrillin gene mutation manifests signs of disease Unaffected individuals may have affected children in some AD diseases Non-Penetrance: a is not affected, but b her child is affected. Why didn't a express condition? For example-person could have died early not giving enough time for phenotype to surface. So you have the mutation but for some reason do not display the phenotype--= NON-PENETRANCE So Penetrance = 7 display condition / 8 affected = 7/8 * 100% Penetrance is bulian= yes or no

Glycoprotein Replacement Therapy(for single enzyme deficiency now, not I-cell)

a) exogenous - M-6-P proteins can be taken up from outside of cell via receptor. b) enter the lysosomal transport pathway c) reconstitute the enzyme & reduce storage problems

Post-translational modifications

a) proteolytic processing at several steps b) hydroxyproline (HP or Hyp) - allows an extra H-bond to form with a peptide carbonyl from another chain to stabilize triple helix. c) hydroxylysine (HK) - site of glycosylation. d) crosslinking of lysines - adds extra strength to fibrils.

BIOSYNTHESIS OF COLLAGEN(steps)

a). mRNA=>Preprocollagen, signal peptide removed during translation. b). Procollagen form has extra amino & carboxyl termini peptides. c). P & K are hydroxylated. d). HydroxyK is glycosylated. e). Prochains associate and carboxyl -termini peptides are disulfide-linked. f). Triple helix formation is initiated at C-terminus and zips up towards N-terminus. g). Molecule is secreted out of cell. h). Amino & carboxyl peptides cleaved to form tropocollagen. i). Self-assembly of many tropocollagens into a 1/4 staggered array. j). Cross-linking of lysines initiated by action of lysyl oxidase - makes allylysine = all. Then Schiff base formation with another K.

Medical Relavence

a. Prevent hemorrhage by forming a hemostatic plug but avoid inappropriate thrombus, which often leads to complications b. Complex cell bio orchestrated by various proteins that elegantly control hemostasis ranging from a bruise to a gash C. acquired defects in any one of steps of hemostatis can signal presence of underlying problems D. many genetic defects can lead to inappropriate clotting or excessive bleeding

Prader-Willi

deletion of paternal 15q11-q13

3. Slow (hour to day time scales)

a. Adjustment of enzyme synthesis rate: - transcriptional (e.g. induction, repression), or - translational (e.g. mRNA stability or access). b. Degradation (e.g. local proteases digest enzyme; send Ubiquitinated enzyme to proteosomes for destruction).

Enzymes & Temperatures

a. All chemical reactions proceed quicker with higher temperature (hypothermia allows tolerance of hypoxia). b. But at a certain point, the enzyme can be denatured as the weak bonds that hold proteins together for most part eventually are broken (hypothermia tolerated better than hyperthermia).

2. Intermediate (minute time scale)

a. Covalent modifications (e.g. phosphorylation by kinase). b. Binding to modulator proteins (e.g. cAMP regulatory subunit). c. Zymogen activation (e.g. clip by a protease to make active form).

Genetic Defects of HB (Decreased Solubility)

a. Crystalline or polymeric Hb (ex. HbS, HbC, HbD, HbE). b. Unstable Hb (ex. Hb Koln).

Noncompetitive (Reversible inhibition)

a. Inhibitor & substrate bind simultaneously to enzyme molecule at different sites and productive catalysis is slowed or prevented (special - allosteric inhibition). b. I works at a different site on enzyme, thus inhibition possible at any [S] (nice choice for a drug). c. Km value same with or without I present (just takes some of the enzyme out of action).

Competitive (Reversible) Inhibition

a. Substrate cannot get to the active site when inhibitor is bound at active site - mutually exclusive binding (inhibitor typically resembles substrate). b. Inhibitor works best at low [S]. c. Inhibitor makes the measured Km value appear higher since more S required for availability to enzyme.

1. Fast (virtually immediate to seconds time scale)

a. Substrate concentration (e.g. reactant build up). b. Allosteric control (e.g. feedback via buildup or loss of a pathway metabolite).

Enzymes & pH

a. The catalytic groups of enzyme and/or groups of the substrate must be in proper protonation state. b. Typically a peak of activity in a certain pH range. c. But at pH extremes, global folding problems or precipitation. (note: if blood greater than 0.5 pH units from pH 7.4, it is fatal).

Chronology from Cut to Clot

a. wound b. platelet adhesion c. platelet activation d. clot initiation e. clot formation f. clot stabilization g. clot retraction

DiGeorge

aka velocardiofacial aka Sphrintzen aka 22q -deletion of 22q11.21-q11.23 -long face -tapering fingers -cleft palate -congental heart defects -seizures -renal anomalies -wide nose -hooding of upper eye lids -bulbous nasal tip -hypernasal voice -immune deficiency -psych illness -autosomal dominant

Intergenerational instability of the CAG repeat

all persons with repeats 40 and > get HD and 97% of pts inherit these alleles

Stage

anatomic extent of the tumor determine by surgery, imaging, pathology

Continuous Phenotypes

are determined by a combination of genes and the environment (colorful fish picture)

Erythrocyte differentiation

basophilic erythroblast => polychromatic erythroblast (last one capable of mitosis) => otochromatic erythroblast

Bullous pephigoid

blistering skin disease due to production of autoantibodies to protein in plaque in hemidesmosomes

angio-

blood vessels or lymphatics

oseo-

bone

Osteoblast

bone forming cells - mesenchymal cells differentiate into osteoblasts - located on surface of bone - synthesize and mineralize bone tissue

limitations of CMA/aCGH

cannot detect balanced rearrrangements -reciprocal translocations -inversions -insertions - does not provide information regarding the location of the additional copy -translocation -marker -tandem duplication can identify CNVs with unclear clinical significance does not identify low level mosiacisn

chondro-

cartilage

Unequal Recombination

causes deletions/duplications CONCEPT: deletions/duplications mediated by segmental duplications ex:crossing over ex:Prader Willi Syndrome & Angelman Syndrome

Post-translational Modification

changes made to a polypeptide chain after translation

Genomic Disorders

chromosomal microdeletion sydromes, contiguous gene syndrome -sub microscopic deletions/duplications of genetic material -usually more than one gene involved -areas of high recombination flanked by and due to unique low copy repeats -following non-homologous recombination caused by misalignment of chromatids during meiosis

Tuberous Sclerosis Complex by Diagnosis complicated

clinically by Variable expression molecularly by Somatic mosaicism & Germline mosaicism

restriction enzyme

cut DNA, thus making it available to rejoin with other DNA to create recombinant DNA

examples of short mutations

cystic fibrosis huntingtons, at least in the example *depending on the number of repeats, could it be a large mutation also?

Two Branches of Mutations

cytogenetic and molecular

Cytoplasm

cytosol and organelles, the site of protein synthesis and intermediary metabolism

Grade

degree of differentiation of the tumor determine by microscopic examination)

Endosome

delivery method after material is taken into the cell via endocytosis Early: Just beneath plasma membrane, proton pump maintains pH 6 Late: Close to Golgi apparatus and nucleus, more acidic than early

Williams syndrome

depressed nasal bridge -large mouth, full lips -hypoplastic nails -prominent earlobes -renal anomalies -joint laxity -hypercalcemia -supravalvular aortic stenosis -iq ranges 41-80 -hoarse voice -friendly -stellate irides -FTT in infancy (failure to thrive) _microdeletion involving elastin gene -autosomal dominant

microarrays

detects small changes not seen via conventional cytogenetic analysis or FISH BUTTTT cannot detect balanced translocations OR low levels of mosaicism

Surgical Pathology

diagnosis of disease by gross examination, histomorphology, immunohistochemistry, and other methods

Cytopathology

diagnosis of disease by microscopic analysis of cells and cellular aggregates

Protein Trafficking

directed movement of proteins within and out of the cell

Squamos

flattened cells

phenylalanine hydroxylase (PAH)

enzyme necessary to metabolize the amino acid phenylalanine (Phe) to the amino acid tyrosine

Balance between risky and beneficial factors (and not one specific trigger) = multifactorial disease

example - skin

Extra-versus intracellular Receptors

extracellular - hydrophilic signal molecule intracellular - hydrophobic signal molecule w/ a carrier protein

lipo-

fat

fibro-

fibroblasts

Molecular pathology

findings from the examination of the DNA and/or RNA of specimens

Histopathology

findings from the microscopic examination of stained tissues

Synthesis of recombinant proteins

for therapies (insulin, growth hormone...)

aneuploidy

having one extra or one less chromosome...

Connexon

hexagonal arrangement of connexins arranged to align with pores of apposing membranes

genomic DNA library

human genomic DNA ---> fragmented and cloned--->??

osteocytes

in lacunae - trapped in lacuna but keep in contact w/ one another

Congenital absence of the vas deferens (CBAVD)

is a condition in which the vasa deferentia, male reproductive organs, fail to form properly prior to birth. It may either be unilateral or bilateral

Bevacizumab

inhibits VEGF, prevents rumors from growing their own blood supply to hopefully starve them

Squamos intracepithelial Lesions

insert picutre

Some connective tissue cells derived from hematopoietic stem cells:

insert slide 21

The four bonds

ionic bond - electrostatic hydrogen bond - sharing a proton (acids=donors, bases=acceptors) hydrophobic force - water wants to "flirt around" van der WALLs force (sum of the attractive or repulsive forces between molecules); dipole forces

perichondrium bone

layer of dense irregular connective tissue that surrounds the cartilage of developing bone

mature bone

lamellar bone

Invasiveness of Cancer

loss of E-cadherin allows the cancer cells to escape to metastasize e-cadherin forces cells to stick together - tumor cells interact w/ benign cells & stroma using heterotypic signaling

Tumor suppressor gene

loss of function when working, these act as the breaks to prevent uncontrolled division

A preganant woman with severe rheumatoid arthritis was treated with d-penicillamine. This drug has an adverse side-effect due to its ability to chelate copper ions. The woman's child is born with lax, soft skin at birth. The physician thinks it is an Ehler-Danlos syndrome. However, within 3 months, the skin gets stronger. What is the most likely enzyme effected in the child?

lysyl oxidase

RT-PCR

mRNA ---> double stranded cDNA (=template for PCR) ------> then can go on to regular PCR advantages: -detection of splice-site mutations -convenient mutation detection (no introns)

cDNA library

mRNA from a particular tissue ---> converted into cDNA and cloned--->??

duplication 22q

mild facial dysmorphism cleft palate congenital heart defect hydronephrosis seizures or infantile spasm intellectual deficits speech delay behavior or psychiatric problems

monosomy

missing a single chromosome

mutagenesis

most arise due to replication errors. errors occur about eery 1 in 1-10 billion bases replicated

Oncogene

normal genes that, when mutated to a more active form, or overexpressed, contribute toward a neoplastic phenotype (gain of function)

Cellularity

normal is about 100-patient's age

Rb

normally prevents cell from progressing from G1 to S, if hyperphosporylated then E2F released and S phase begins

Sporadic mutation of Rb

no inheritance, must have 2 random mutations to become cancerous

PKU (Clinical)

on newborn screen if positive, immediate clinic appt to confirm if affected, diet for life ideally, Phe levels <6 mg/dL where classically affected usually >20 "benign" hyperphe high levels phe is brain toxin maternal PKU serious teratogenic risk

Follicular lymphoma

oncogene: BCL2 t(14;18) causes overexpression of BCL2 and resistance to apoptosis

Breast Carcinoma

oncogene: HER2 amplification of HER2, increased signaling, proliferation Amplified--then able to use trastazumab (targeted therapy)

endocrine

over large distance

paracrine

over short distance

numerical cytogenetic mutation

polyploidy anueploidy

High-grade

poorly differentiated (does not look like tissue of origin (anaplastic))

Basal Lamina

produced by the epithelium and cells of the underlying connective tissue

bone lining cells

quiescent osteoblasts of the endosteum and peristeum

trisomy

single extra chromosome

rhabdomyo

skeletal muscle

leiomyo-

smooth muscle

Cell junctions

specialized proteins that provide cell-to-cell adhesion and communication, and cell-to-matrix adhesion

DNA repair pathways

specific pathways for specific lesions

exome

that part of the genome that is responsible for coding.

cytogenetic

the branch of biology that studies the cellular aspects of heredity (especially the chromosomes)

Gain of function

the mutant protein gains (unexpectedly) a new function - eg. a signalling protein is always 'on' - eg. achondroplasia (FGFR3)

Dominant negative effect

the mutant protein interferes with the function of the normal proteins - eg. in osteogenesis imperfecta (COL1A1 and COL1A2) Dominant negative effect-interfering with normal function of proteins

Loss-of-function effect

the mutant protein is not available to the extent the (developing) organism requires - eg. heterozygous mutation in TGFBR1 or TGFBR2 cause Loeys-Dietz syndrome, a Marfan-like disorder

Mosaicism

the presence of two or more populations of cells with different genotypes in one individual who has developed from a single fertilized egg

Anueploidy

total chromosome count is not a multiple of n

Mesenchymal tumor

tumor affecting connective tissue

T_N_M_

tumor designation T=tumor size/extent N=nodal status M=metastasis

Addition of peptides groups

ubiquitination

Protein Clearance

uptake of secreted proteins from extracellular location and degradation within the cell

low-grade

well-differentiated (looks like tissue of origin)

metaphase

what phase for staining prep

immature bone

woven bone

Cystic Fibrosis (mutation0

ΔF508 - most common mutation, defect in processing R117H mutation is variable area within CFTR consisting of multiple Ts (5, 7, or 9) R117H in cis with 5T, deleterious R117H in cis with 7T, possibly mild effects R117H in cis with 9T, probably not CF effects except CBAVD

KRAS mutations are found in many colon cancers

• "Point mutation of RAS family genes is the single most common abnormality of dominant oncogenes in human tumors. Approximately 15% to 20% of all human tumors contain mutated versions of RAS proteins." • Colon cancer:50% have point mutations • KRAS mutations predict decreased responsiveness to monoclonalAb therapy against epidermal growth factor receptor (EGFR) (e.g.,cetuximab,panitumumab)

About half of melanomas have BRAF mutations

• Molecular testing can be done on formalin-fixed specimens of melanoma • Melanomas with BRAFV600E (valine to glutamate at amino acid position 600,i.e.,a missense mutation) respond to BRAF kinase inhibitor (vemurafenib)

Strategies to Prevent Death from Pneumococcal Sepsis

- Start penicillin prophylaxis in early infancy and continue until 5 years of age - Special immunizations: Prevnar (now 13-valent at 2, 4 and 6 and 15 months) Pneumovax (23 valent) at 2 and 5 years Meningococcal vaccine Flu shot annually - Prompt evaluation for fever >101 F

Down Syndrome

- a particular combination of phenotypic features that includes mental retardation and characteristic facies (Down, 1866) - caused by the presence of an extra copy of chromosome 21 as a free chromosome as a Robertsonian translocation, or as a reciprocal translocation involving part of chromosome 21

Cystic Fibrosis:The Basic Defect

- abnormal CFTR & epithelial ion movement - abnormal movement of ions across the CFTR protein channel - causes build up of mucus across lungs

Therapy for CF for mutated CFTR

- airway clearance to remove airway mucus - inhaled hypertonic saline & mannitol to rehydrate airway lining fluid - Kalydeco to re-establish Cl- transport via CFTR protein - RESULTS: normal function of cilia & mucus => relieves physical airway obstruction & clears bacteria from airway

Therapy for bacteria in airways

- antibiotics (mostly inhaled), reduce bacterial growth - inhaled - tobramycin, azteronam, possibly ciprofloxacin, levofloxacin & amikacin - Azithromycin inhibits biofilm formation - RESULT: reduced bacterial load => less infection & inflammation

transcranial doppler screening

- are tests that measures the velocity of blood flow through the brain's blood vessels. - considered relatively inexpensive - Who do we screen? Hgb SS & Hgb S beta0thal Aged 2-16 Annually

Mutated CFTR in CF

- cell surface protein (CFTR) is either missing or has a very low level or not working - w/ no or little Cl- at cell surface, there is little NaCl & thus a low amount of water - w/ little water to float upon, the airway mucus doesn't move - when bacteria land in the mucus, they are not carried "up & out" and become a chronic airway infection

Cystic Fibrosis

- develops due to a defect in either the amount of protein at the cell surface, decreased activity of the protein, or combination of both - some CFTR surface proteins may be less active => leads to milder disease (classes III-V) - no CFTR protein => more severe disease (I-II)

Therapy for WBCs arriving to infected airways

- ibuprofen may inhibit WBC (neutrophil) influx to the area - polmozyme (DNAse) breaks down DNA matrixes - RESULT: w/ less WBCs, mucus is thinner & easier to clear => less inflammatory airway damage=more slowly developing bronchiectasis

CF: Pathophysiology

- livery and billiary tract disease - pancreatic insufficiency - CF-related diabetes - meconium ileus - Intestinal obstruction (DIOS) - nasal polyps - sinus disease - acute & chronic bronchitis - emphysema - male infertility (azoospermia) - electrolye abnormalities - BOTTOM: BAD LUNGS

Treatment of Vaso-occlusive Pain

- local heat- no ice packs - hydration - combination medications work better (NSAID+narcotic +/- tylenol) - pain medications should be prescribed on a regular dosing schedule - not "as needed" - Always consider other causes (other diseases or other compications of sickle cell) - Nsaids- ibuprrofen, narcotics - codeine, morphine

CF: Sweat levels

- normal sweat: < 40 mM NaCl - CF sweat: > 60 mM NaCl

Sickle Cell and Spleen Functional Asplenia

- pre-pencillin era: incidence of pneumococcal sepsis-increased by 200-400 fold in children w/ sickle cell disease

Recombination and the concept of linkage

- Recombination occurs approx. 1 / chromosome arm - Loci on different chromosomes or widely separated on the same chromosome show independent assortment - Closely positioned loci are less likely to be separated by recombination and are said to be linked (genetic linkage)

Oklahoma Pediatric Sickle Cell Program

- Set up in 1993: 1 year after hemoglobinopathies were added to newborn screening - Dual mandate: Track all babies diagnosed by newborn screening Provide health maintenance care to all children with sickle cell disease from birth to 21 years

Stroke

- 10% of children with sickle cell disease will have a stroke during childhood - Almost all pts have SS or S beta0 thalassemia - Significant decreases in IQ and quality of life - Without therapy, incidence of recurrence is >50%

Newborn Screening in Oklahoma

- 1963: PKU 1979: Hypothyroidism 1991: Galactosemia Hemoglobinopathies 2004-2006: CF, CAH, MCAD

Recurrence risk for Trisomy 21

- Affected by maternal age & parental germline mosaicism - After 1 DS pregnancy, generally quoted as 1-2%, but higher if age-based risk is higher - After 2 DS pregnancies, may be as high as 10%

Cytogenetics (cont'd)

- By 3rd month of fetal development, the oogonia of the female embryo have begun to develop into primary oocytes, reaching the prophase of meiosis I before birth - The preponderance of meiosis I non-disjunction is likely to due prolonged meiotic arrest in prophase I - On reaching sexual maturity, each individual follicle matures, and ovulation occurs; the oocyte completes meiosis I, proceeding to metaphase in meiosis II - Meiosis II is only completed if fertilization occurs

Normal Defensive pathway of protecting airways from infection

- CFTR moves chloride ions from inside the cell to the cell surface

Sickle Cell Pain

- Caused by ischemia or infarction of bone - May occur at any site - Often has a typical location - Pt usually has no physical findings other than pain. Dactylitis in infancy is the exception. - No lab test will tell if the pt is having pain: Hgb, bilirubin, % of sickled cells on smear do not change from baseline values in most vaso-occlusive crises. Xray and MRI often not helpful

Cardiac Complications

- Congenital structural heart disease - Atrioventricular septal defects (60% of CHD) Ventricular septal defect (VSD) Atrial septal defect Patent ductus arteriosus Other complex heart disease, eg: Tetralogy of Fallot & Hypoplastic left heart

Duchenne Muscular Dystrophy

- DMD - x -linked recessive - Female in 3rd generation is an obligate carrier for the mutation (50% risk of being a carrier) - for her child (1/4) and for her son (50%) - we can use ck (creatine kinase) measurements to decipher chances of offspring (off III-2) getting disease

Other causes of down syndrome

- DS can also occur as a result of a mitotic non-disjunction event after zygote formation - This may result in clinically significant mosaicism, often with a milder phenotype

Other Causes of Down Syndrome

- DS can also occur when one parent carries a translocation involving chromosome 21 - This occurs in about 5% of cases - In that case, the risk to the offspring is dependent on the gender of the carrier parent - If father carrier: <1% - If mother carrier: 10-15% - Situation is different if t(21:21) in one parent

TCD Interpretation

- Flow velocity <170cm/sec=low risk - Flow velocity 170-200 cm/sec=conditional, may be at increased risk Flow velocity of 200cm/sec=at risk to develop stroke (10% per year)

Biochem of Sickle Cell Review

- Hemoglobin consists of two alpha and two beta globin chains - Mutation of one nucleotide leads to substitution of Glu for Val at the 6th position on beta globin chain on chromosome 11 - Change in surface charge of Hgb - Change in the alignment of Hgb molecules / polymerization - Change in shape of RBC under certain conditions

Sickle Cell Laboratory Features

- Hemolytic Anemia: a. Low baseline Hgb b. Elevated reticulocyte count c. Hyperbilirubinemia d. Increased LDH (lactate dehydrogenase) - Elevated WBC (decreased margination) - Elevated platelets (decreased splenic function)

Hydroxyurea Beneficial effects

- Increase in Hgb F=>interferes with polymerization of Hgb S & sickling of RBCs - Decreases adhesiveness and endothelial damage - Macrocytosis (enlargement of red blood cells with near-constant hemoglobin concentration) & increased hydration reduces hemolysis and sickling - Local release of nitric oxide results in vasodilation - Decrease in inflammation/tissue damage - Decrease in WBC and platelet count - Decrease in cytokine release

Factors Favoring Polymerization

- Increased temperature - Acidosis - Dehydration - Deoxygenation of RBC (i.e. distance from pulmonary bed) - Absence of other normal hemoglobins (e.g. Hgb A, Hgb F)

Other therapies for CF

- Nutritional support (High calorie, high protein diet, often special supplements, sometime enteral feeding) - Pancreatic enzyme supplementation - Vitamin supplements with altered fat-soluble vitamins - Close relationship between nutritional status & pulmonary function - With G551D mutation - correction of the functional cellular defect w/ Kalydeco

CF: Diagnosis

- One or more characteristic CF phenotypic features: - chronic sino-pulmonary disease - GI and/or nutritional abnormalities - salt loss syndromes - obstructive azoospermia - sibling with CF - Plus laboratory evidence of CFTR abnormality - ~52% respiratory symptoms

Examples of "over-exposed" Genes

- Over-expression of superoxide dismutase (SOD1) may cause premature aging and decreased function of the immune system - COL6A1 over-expression may be the causative of heart defects - DYRK over-expression may cause the mental retardation seen in DS - CRYA1 over-expression may be the cause of cataracts

Prenatal Screening and Diagnosis

- The American College of Medical Genetics (ACMG) has issued technical standards and guidelines for prenatal screening for Down syndrome - The guidelines distinguishes screening from diagnostic testing - The distinction is important, because the goals and expectations differ for clinical sensitivity and specificity, costs, and acceptable level of invasiveness..

Positional cloning of genes is done without prior knowledge of the defective / deficient protein

- The disease trait is first mapped to a specific chromosomal region: cytogenetic abnormality (deletion, translocation) & genetic linkage analysis (family studies) - The defective gene is then identified in this chromosomal sub-region using DNA technology - Finally, the deficient/defective protein is identified and expressed using the newly identified gene (cDNA) sequence - Gene identification nowadays relied heavily on (combination of) linkage analysis and homozygosity mapping - being replaced by NGS

Duchenne Muscular Dystrophy

- WHEN THERE IS NO AFFECTED PATIENT PRESENT, WE CAN FOLLOW THE MARKERS - II-2 is an obligate carrier. - III-4 must have inherited the normal allele - III-3 must have inherited the other allele, carrying the DMD mutation - 11 d 2 seems to be the disease-carrying allele

Management/Intervention (Abstract)

Evaluate for congenital heart disease Evaluate for hearing loss (through NBS) Evaluate for hypothyroidism (through NBS) Be aware of gastrointestinal complications Be aware of hematologic complications

Eye & ENT complications

Eye: - Myopia - Cataract ENT: - Recurrent otitis media (horizontal Eustachian tubes) - Conductive hearing loss - Protruding tongue, likely due to small mid-face

Hematological Complications

Transient myeloproliferative disorder Leukemia - 200 to 400 times more frequently in the Down syndrome (Zipursky et al. 1987) - Acute myeloid leukemia (AML) outcome generally favorable associated with somatic mutation in GATA1 (Xchr) - Acute lymphoblastic leukemia outcome not as favorable